Generation and maintenance of stem cells

ABSTRACT

The present invention provides for the generation and maintenance of pluripotent cells by culturing the cells in the presence of an ALK5 inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/490,433, filed Sep. 18, 2014, which is a continuation of U.S.application Ser. No. 13/140,108, filed Sep. 1, 2011, issued as U.S. Pat.No. 8,906,677, which is a U.S. National Phase application under 35U.S.C. §371 of International Application No. PCT/US2009/068274, filedDec. 16, 2009, which claims priority to U.S. Provisional Application No.61/138,407, filed Dec. 17, 2008, the disclosures of which areincorporated by reference herein.

BACKGROUND OF THE INVENTION

Although embryonic stem cells (ESCs) have been established from micesince 1981, attempts to derive their counterparts from various othermammals, including rats, have not succeeded. Recently, pluripotent stemcells were derived from the post-implantation egg cylinder stageEpiblasts of mouse and rat (Brons et al., Nature 448, 191-195 (2007);Tesar et al., Nature 448, 196-199 (2007)). These novel stem cells werenamed Epiblast stem cells (EpiSCs). EpiSCs seem to correspond veryclosely to human embryonic stem cells (hESCs) in the colony morphologyand culture/signaling requirements for maintaining pluripotency, butexhibit a range of significant phenotypic and signaling responsedifferences from the mouse ES cells (mESCs).

Leukemia inhibitory factor (LIF) is essential for maintaining thepluripotency of mESCs in the presence of serum through JAK-STAT3 pathway(Niwa et al., Genes Dev 12, 2048-2060 (1998)). However, in serum-freemedium, BMP4 is also required, together with LIF, to sustain mESCself-renewal by inducing inhibitor of differentiation (Id) proteinexpression (Ying et al., Cell 115, 281-292 (2003)) and inhibiting ERKactivation (Qi et al., Proc Natl Acad Sci USA 101, 6027-6032 (2005)). Incontrast to mESCs, LIF cannot support EpiSCs/hESCs, which typicallyrequire basic fibroblast growth factor (bFGF)/Activin A for long termself-renewal. Undifferentiated hESCs display high-level basal activityof ERK through bFGF signaling (Dvorak et al., Stem Cells 23, 1200-1211(2005)). BMP4 doesn't support EpiSC/hESC self-renewal either, butinstead induces EpiSC/hESC to differentiate into trophoblasts orprimitive endoderm (Brons et al., Nature 448, 191-195 (2007); Tesar etal., Nature 448, 196-199 (2007); Xu et al., Nat Biotechnol 20, 1261-1264(2002)). In addition to bFGF, Activin A/Nodal signaling has been shownto support the undifferentiated state of hESCs/EpiSCs (Brons et al.,Nature 448, 191-195 (2007); Sato et al., Dev Biol 260, 404-413 (2003);Tesar et al., Nature 448, 196-199 (2007)), while is dispensable formESCs. These results strongly support the notion that EpiSCs and hESCsare intrinsically similar and raise an attractive hypothesis that mESCsand EpiSCs/hESCs represent two distinct pluripotent states: themESC-like state representing the pre-implantation inner cell mass (ICM)and EpiSC-like state representing later Epiblast cells, respectively.

mESCs can be usually derived from certain mouse strains using feederlayer based cell culture conditions (Martin, G. R., Proc Natl Acad SciUSA 78, 7634-7638 (1981)). However, it has been proven difficult toderive authentic ES cells from rats under similar conditions.Establishments of rat ESC-like cells have been reported (Demers et al.,Cloning Stem Cells 9, 512-522 (2007); Ruhnke et al., Stem Cells 21,428-436 (2003); Schulze et al., Methods Mol Biol 329, 45-58 (2006); Uedaet al., PLoS ONE 3, e2800 (2008)), but these cells either could not bestably maintained or lacked true in vivo pluripotency (e.g. fail to formteratoma or no/little contribution to chimerism). Similarly, although(in vitro) pluripotent rat EpiSCs had been derived, both rat and mouseEpiSCs show little or no ability to be reincorporated into thepre-implantation embryo and contribute to chimaeras (Brons et al.,Nature 448, 191-195 (2007); Tesar et al., Nature 448, 196-199 (2007)).

Recently, induced pluripotent stem cells (iPSCs) generated from bothmouse and human somatic cells by defined genetic transduction haveattracted enormous interests (Dimos et al., Science 321, 1218-1221(2008); Han, J., and Sidhu, K. S. Curr Stem Cell Res Ther 3, 66-74(2008); Takahashi et al., Cell 131, 861-872 (2007); Takahashi, K., andYamanaka, S., Cell 126, 663-676 (2006); Yu et al., Science 318,1917-1920 (2007)).

BRIEF SUMMARY OF THE INVENTION

The present invention provides for methods of culturing pluripotentcells through at least one cell divisional. In some embodiments, themethods comprise culturing pluripotent animal cells in the presence of asufficient amount of:

-   a. an ALK5 inhibitor (or other TGFβ/activin pathway inhibitor), and-   b. a second compound selected from one or more of a MEK inhibitor,    an Erk inhibitor, a p38 inhibitor, and an FGF receptor inhibitor;    and-   c. sufficient nutrients for a sufficient time, to allow for at least    one cell division while maintaining cell pluripotency.

In some embodiments, the culturing step further comprises culturing thecells in the presence of an amount of a GSK3β inhibitor. In someembodiments, the GSK3β inhibitor is CHIR99021.

In some embodiments, the second compound is a MEK inhibitor. In someembodiments, the MEK inhibitor is PD0325901.

In some embodiments, the second compound is a Erk inhibitor.

In some embodiments, the culturing step is performed in the furtherpresence of Leukemia inhibiting factor (LIF).

In some embodiments, the ALK5 inhibitor is A-83-01. In some embodiments,the ALK5 inhibitor is SB431542.

In some embodiments, the pluripotent cells are cultured through at leastfive cell divisions while maintaining cell pluripotency.

In some embodiments, the method further comprises introducing aheterologous nucleic acid into the pluripotent cells and culturing theresulting cells to allow for at least one additional cell divisionalwhile maintaining pluripotency. In some embodiments, a heterologousnucleic acid is introduced into animal cells, then induced topluripotency, and then submitted to the culturing step.

In some embodiments, the cell is a rat or human cell. In someembodiments, the cell is a primate, ovine, bovine, feline, canine, orporcine cell.

In some embodiments, the pluripotent cells are embryonic stem cells. Insome embodiments, the pluripotent cells are induced pluripotent stemcells.

In some embodiments, the cells are non-human animal cells and the methodfurther comprises introducing the pluripotent cells into a blastocyst,wherein the blastocyst is from the same species of animal as the cells,and introducing the blastocyst into the uterus of an animal of the samespecies. In some embodiments, the method comprises selecting chimericprogeny of the animal based on the presence of a nucleic acid from thepluripotent cells.

The present invention also provides for cultures of pluripotentmammalian cells. In some embodiments, the cultures comprise a sufficientamount of:

-   a. an ALK5 inhibitor (or other TGFβ/activin pathway inhibitor), and-   b. a second compound selected from one or more of a MEK inhibitor,    an Erk inhibitor, a p38 inhibitor, and an FGF receptor inhibitor;-   to allow for at least one cell division while maintaining cell    pluripotency.

In some embodiments, the cultures further comprise LIF.

In some embodiments, the cultures further comprise an amount of a GSK3βinhibitor. In some embodiments, the GSK3β inhibitor is CHIR99021.

In some embodiments, the second compound is a MEK inhibitor. In someembodiments, the MEK inhibitor is PD0325901.

In some embodiments, the second compound is a Erk inhibitor. In someembodiments, the ALK5 inhibitor is A-83-01. In some embodiments, theALK5 inhibitor is SB431542.

In some embodiments, the cell is a rat or human cell. In someembodiments, the cell is a primate, ovine, bovine, feline, canine, orporcine cell. In some embodiments, the cells are induced pluripotentstem cells or embryonic stem cells.

The present invention also provides a cell culture medium. In someembodiments, the medium comprises a sufficient amount of:

-   a. an ALK5 inhibitor (or other TGFβ/activin pathway inhibitor), and-   b. a second compound selected from one or more of a MEK inhibitor,    an Erk inhibitor, a p38 inhibitor, and an FGF receptor inhibitor to    allow for at least one cell division while maintaining cell    pluripotency when pluripotent cells are cultured in the medium.

In some embodiments, the medium further comprises LIF.

In some embodiments, the medium further comprises an amount of a GSK3βinhibitor. In some embodiments, the GSK3β inhibitor is CHIR99021.

In some embodiments, the second compound is a MEK inhibitor. In someembodiments, the MEK inhibitor is PD0325901.

In some embodiments, the second compound is a Erk inhibitor. In someembodiments, the ALK5 inhibitor is A-83-01. In some embodiments, theALK5 inhibitor is SB431542.

In some embodiments, the medium is in a pre-packaged, seal container. Insome embodiments, the medium comprises DMEM or other media compatiblefor growing human, rat, mouse or other animal cells.

The present invention also provides isolated pluripotent animal cellsthat replicates and maintains pluripotency in the presence of leukemiainhibitory factor (LIF) and bone morphogenic protein (BMP), or underinhibition of the TGFβ and activin signaling pathway, inhibition of theMAPK signaling pathway, and optionally inhibition of the FGF pathway. Insome embodiments, the isolated pluripotent animal cell is not a murineembryonic stem cell (mESC). In some embodiments, the cell is a humancell. In some embodiments, the cell is a human embryonic stem cell. Insome embodiments, the cell is a human iPS cell. In some embodiments, thecell is a rat cell. In some embodiments, the cell is a rat embryonicstem cell. In some embodiments, the cell is a rat iPS cell. In someembodiments, the cell maintains pluripotency under inhibition of ALK5and MEK. In some embodiments, the cell comprises a heterologousexpression cassette, including but not limited to an expression cassetteencoding a selectable or detectable marker (e.g., alkaline phosphatase).

The isolated pluripotent cell of the present invention expresses ahigher level of E-cadherin as compared to conventionally-cultured hESCs,Epiblast stem cells and human induced pluripotent cells. For example,the isolated pluripotent animal cell expresses a 2-fold higher level ofE-cadherin as compared to conventionally-cultured hESCs, EpiSCs andhuman induced pluripotent cells. In some embodiments, the isolatedpluripotent animal cell expresses a higher level of markers as comparedto conventionally-cultured hESCs, Epiblast stem cells and human inducedpluripotent cells, wherein the markers include Gbx2, Dppa3, Klf4, andRex1.

In some embodiments, the isolated pluripotent cell of the presentinvention is cultured in the presence of an ALK5 inhibitor, a secondcompound selected from a MEK inhibitor, an Erk inhibitor, a p38inhibitor, and an FGF receptor inhibitor. In some embodiments, theisolated pluripotent cell of the present invention is obtained orobtainable by culturing a cell in the presence of an ALK5 inhibitor, anda second compound selected from one or more of a MEK inhibitor, an Erkinhibitor, a p38 inhibitor, and an FGF receptor inhibitor. For example,the isolated pluripotent cell of the present invention is obtained orobtainable by culturing conventionally-cultured hESCs, EpiSCs, rat ESCs,or primate ESCs.

The present invention also provides methods of increasing thepluripotency of a partially pluripotent mammalian cell to a more fullypluripotent cell. In some embodiments, the methods comprise,

-   (a) contacting the partially pluripotent cell with an epigenetic    modifier selected from a histone deacetylase inhibitor, an inhibitor    of histone H3K4 demethylation or an activator of H3K4 methylation;-   (b) after step (a) culturing the cell with two or more of (i) an    ALK5 inhibitor, (ii) a MEK inhibitor, an Erk inhibitor, or a p38    inhibitor, and (iii) an FGF receptor inhibitor and in the absence of    the epigenetic modifier, thereby generating the more fully    pluripotent cell as compared to the partially pluripotent mammalian    cell.

In some embodiments, the methods further comprise

-   (c) culturing the partially pluripotent mammalian cell after    step (b) with (i) an ALK5 inhibitor, (ii) a MEK inhibitor, an Erk    inhibitor, or a p38 inhibitor, and (iii) an FGF receptor inhibitor    and (iv) a GSK3 inhibitor.

In some embodiments, culturing steps (a) and/or (b) and/or (c) furthercomprises culturing the partially pluripotent mammalian cell in thepresence of Leukemia inhibitory factor (LIF).

In some embodiments, the partially pluripotent cell is an Epiblast stemcell.

In some embodiments, the partially pluripotent cell does not express atleast one marker selected from the group consisting of Oct4, Nanog,SSEA-1, and REX-1 and the more fully pluripotent cell expresses one ormore or all of the markers.

In some embodiments, the partially pluripotent cell does not expressALP-1 and the more fully pluripotent cell expresses ALP-1.

In some embodiments, the epigenetic modifier is valproic acid orparnate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1K. mESC-like riPSCs could be generated from rat WB-F344 cellsafter transduced with Oct4, Sox2 and Klf4 by retroviruses andcaptured/maintained under combination of LIF, 0.5 μM PD0325901, 0.5 μMA-83-01 and 3 μM CHIR99021. ESC-like colonies were observed 10 daysafter transduction (A), but could not be maintained in the conventionalmESC culture condition (B). In the presence of 0.5 μM PD0325901 and 3 μMCHIR99021, riPSCs can be short-term maintained in culture but showextensive spontaneous differentiation (C). With the combination of 0.5μM PD0325901, 3 μM CHIR99021, and 0.5 μM A-83-01, riPSCs can long-termand homogenously self-renew (D), and form mESC-like domed colonies inculture (E). Immunocytochemistry revealed that riPSCs express typicalmESC markers, such as Oct4 (F), Sox2 (G), SSEA-1 (H, Green) and Nanog(H, red). RT-PCR analysis of four clonal riPSC lines confirmed theexpression of endogenous typical pluripotency markers (I), but thevirally transduced genes were largely silenced. Oct4 promoter of riPSCclones exhibited a demethylation pattern and is distinct from that ofthe parental WB-F344 cells (J). Karyotyping analysis showed thechromosome number of riPSCs was 42 (K).

FIG. 2A-2I. riPSCs have pluripotent developmental potential in vitro andin vivo. Immunostaining showed riPSCs could differentiate into endoderm(Albumin and Pdx1) (A and B), neuroectoderm (βIII-tubulin, Tuj1) (C) andmesoderm (Brachyury) (D) derivatives in vitro. Also, riPSCs can formteratoma in SCID mice, which consisted of all three germ layers (E-H).In addition, after injected into Brown-Norway rat blastocysts, riPSCswith WB F344 background were capable of producing chimera rats (I).Relative magnification: A-E (100×), F-H and J-Q (200×).

FIG. 3A-3L. The generation of novel “mESC-like” hiPSCs. IMR90 humanfibroblasts were transduced with Oct4, Sox2, Nanog, and Lin28 bylentiviruses. The hiPSC colonies were observed three weeks aftertransduction (A), picked up at the fourth week after transduction andwere stably maintained under the cocktail of hLIF, 0.5 μM PD0325901, 0.5μM A-83-01, and 3 μM CHIR99021. Such hiPSCs formed domed coloniessimilar to mESCs (B). Under such conditions, hiPSCs were positive to ALP(C) and other typical pluripotency markers (D˜I). RT-PCR analysis offour clonal hiPSC lines confirmed the expression of endogenouspluripotency genes (J), but the virally transduced genes were largelysilenced. Oct4 promoter of hiPSCs clones exhibited a demethylationpattern similar to conventionally-cultured human ES cells, but isdistinct from that of the parental IMR90 fibroblasts (K). A karyotypeanalysis of hiPSCs is provided (L).

FIG. 4A-4E. Immunostaining showed that the hiPSCs could effectivelydifferentiate into endoderm (Albumin) (A), neuroectoderm (βIII-tubulin,Tuj1) (B) and mesoderm (Brachyury) (C) derivatives in vitro. Aftertransplanted into the SCID mice, hiPSCs could form teratoma, whichconsisted of all three germ layers including neuroepithelium-likestructure (ectoderm) (D), tube-like structure (endoderm) (D),cartilage-like structure (mesoderm) (E). Relative magnification: A(100×), B˜E (200×).

FIG. 5A-5B. The effects of different small molecule combinations onmaintaining the pluripotency of riPSCs in culture. riPSCs weretrypsinized into single cells and seeded into 6-well plate at thedensity of 103 cells per well and treated with different inhibitorcombinations. Five days latter, ALP staining was performed to visualizethe riPSC colonies (A). The ALP positive colonies for each conditionwere counted from ten random 40× visual fields and the relative colonynumber was summarized (B).

FIG. 6A-6F. EpiSCs differentiate in mESC growth conditions and do notreadily convert to ICM/mESC-like state. (A) Murine ESCs R1 grew ascompact and domed colonies in conventional mESC growth mediumsupplemented with LIF, and the colonies showed positive ALP activity(top left). EpiSCs grew as large and flat colonies in conventional hESCculture medium supplemented with bFGF, and the colonies showed negativeALP activity (top right). EpiSCs differentiated in conventional mESCgrowth medium supplemented with LIF (bottom left); EpiSCs differentiatedin conventional mESC growth medium supplemented with LIF and 0.5 μM MEKinhibitor PD0325901, 0.1 μM FGFR inhibitor PD173074 and 3 μM GSK3inhibitor CHIR99021 (m/MFGi) (bottom right). (B) Schematic for thegeneration of converted cells. EpiSCs were trypsinized to single cells,and plated on feeder cells under the mESC self-renewal condition withsupplements of the indicated chemical compounds for about 4 days toinduce conversion, followed by another 4 days of selection. The culturewas subsequently replated and further selected and expanded for anothertwo weeks, during which time stable clones were picked. (C) Inhibitionof TGFβ signaling by a selective ALK4/5/7 inhibitor A-83-01 (0.5 μM)induced EpiSCs to form more compact and domed colonies that express ALP.(D) These colonies could be further stably expanded in mESC growthmedium supplemented with LIF and 0.5 μM A-83-01, 0.5 μM PD0325901, 0.1μM PD173074 and 3 μM CHIR99021 (mAMFGi). (E) LSD inhibitor parnateinduced EpiSCs to form more compact and domed colonies that express ALP.These colonies could be further stably expanded in mMEGi or (F) mAMFGiconditions. Note the mESC-like domed colonies and positive ALPactivities. Scale bar, 50 μm.

FIG. 7A-7E. EpiSCs convert to ICM/mESC-like state by treatment withparnate and inhibitors of ALK4/5/7, MEK, FGFR and GSK3. (A) Efficiencyin producing chimerism from three types of compound-treated cells. (B)Stable mESC-like cells converted from EpiSCs by the parnate/mAMFGicondition contributed to chimerism in adult mice after aggregatedembryos were transplanted into pseudo-pregnant mice. The Agouti coatcolor originated from Parnate/mAMFGi cells. (C) PCR genotyping for thepresence of GFP integration in multiple adult tissues (D) An E13.5embryo was examined by fluorescence for contribution from theparnate/mAMFGi cells that were labeled with GFP, and GFP-positive cellswere observed in multiple tissues of the embryo (higher magnificationpictures are shown in FIG. 10A). (E) GFP/SSEA-1 double positive cells inthe gonad were isolated by FACS and examined by real-time PCR forgermline markers. The results demonstrated the specific expression ofgermline markers Blimp1 and Stella in the Parnate/mAMFGicells-contributed germline lineage. Bar: ±STDV.

FIG. 8A-8E. Molecular characterizations of the converted Parnate/mAMFGicells. (A) Immunocytochemistry showed homogeneous expression ofpluripotency markers, Oct4 (Green), Nanog (Red), and SSEA-1 (Red) inParnate/mAMFGi cells. (B) Expression of specific ICM marker genes(Rex-1, Pecam1, Dax 1, Dppa5, Esrrb, Fgf4, and Fbxo15), germlinecompetence associated marker genes (Stella and Stra8), and Epiblast gene(fgf5) in mESCs, EpiSCs, and parnate/mAMFGi cells were analyzed bysemi-quantitative RT-PCR. GADPH was used as a control. (C) Transcriptomeanalysis of mESCs, EpiSCs, and parnate/mAMFGi cells showed thatParnate/mAMFGi cells are much more similar to mESCs than EpiSCs. Twobiological replicates were used for all three cell types. (D)Methylation analysis of Stella and Fgf4 promoters by bisulfate genomicsequencing. Open and closed circles indicate unmethylated and methylatedCpGs, respectively. (E) ChIP-QPCR analysis of the indicated histonemodifications in the stella locus in various cells. Genomic DNAs wereimmunoprecipitated from feeder-free cultured EpiSCs, R1-mESCs, andParnate/mAMFGi cells with antibodies as indicated, followed by Q-PCRanalysis using a primer set specific to the endogenous genomic locusencoding Stella. Levels of histone modifications were represented aspercentage of input. IgG served as no-antibody control.

FIG. 9A-9D. Functional characterizations of the converted Parnate/mAMFGicells. (A) Parnate/mAMFGi cells have similar growth rate as mESCs.R1-mESCs and Parnate/mAMEGi cells were passaged every 3 days, and cellnumber was counted every 24 hr. (B) Parnate/mAMFGi cells can effectivelydifferentiate in vitro into cells in the three germ layers, includingcharacteristic neuronal cells (βIII-tubulin and MAP2ab positive),cardiomyocytes (cardiac troponin and MHC positive), and endoderm cells(Sox17 or Albumin positive). Nuclei were stained with DAPI. (C) BMP4 hasdifferential effect on induction of mesoderm marker (Brachyury),trophoblast marker (Cdx2), and primitive endoderm marker (Gata6)expression in EpiSCs, mESCs, and parnate/mAMFGi cells. (D) Directedstep-wise cardiomyocyte differentiation under a monolayer and chemicallydefined condition demonstrated that Parnate/mAMFGi cells share similardifferentiation response as R1-mESCs, and are different from EpiSCs.Cells were characterized with CT3 staining and beating phenotype.

FIG. 10A-10B. Parnate/mAMFGi cells contributed to chimeric miceefficiently. (A) Tissues from chimeric embryos. GFP positive cellscontributed from Parnate/mAMFGi cells were observed in gonad, brain,heart, intestine, lung, and kidney. (B) GFP genotyping of chimeric adultmice. Five mice were randomly picked, and GFP integration in fivedifferent tissues, namely heart, lung, liver, brain, and spleen, wereanalyzed by genomic PCR. Positive detection of GFP integration in allfive tissues of 2 adult mice, in four tissues of 3 mice, confirmed thatparnate/mAMFGi cells could contribute to the three germ layers(mesoderm, endoderm and ectoderm) in vivo.

FIG. 11A-11B. Homogenous expression of pluripotency markers in convertedparnate/mAMFGi cells under feeder cells or feeder-free cultureconditions. (A) The parnate/mAMFGi cells were labeled with GFP, and werepropagated on feeders. Immunostaining results showed homogeneousexpression of GFP and pluripotency-markers Oct4 (Red), Nanog (Red), andSSEA-1 (Red). (B) Left panel: Undifferentiated Oct4-positive coloniesdeveloped from single parnate/mAMFGi cells as efficiently as from singleOG2-ES cells. The colonies were expanded for several passages aftersingle cell seeding in a feeder-free and N2B27-chemically definedcondition. Scale bar, 50 μm. Right panel: Parnate/mAMFGi cellsdifferentiated and lost Oct4 expression in the absence of LIF.Parnate/mAMFGi cells were expanded after single cells seeding in afeeder-free and N2B27-chemically defined condition; the growth factorsupplement was indicated.

FIG. 12. Expression of STELLA was detected in converted parnate/mAMFGicells and R1-mESC cells, but not in EpiSCs. Immunostaining resultsshowed the expression of STELLA, and DAPI.

DEFINITIONS

The term “pluripotent” or “pluripotency” refers to cells with theability to give rise to progeny that can undergo differentiation, underthe appropriate conditions, into cell types that collectivelydemonstrate characteristics associated with cell lineages from all ofthe three germinal layers (endoderm, mesoderm, and ectoderm).Pluripotent stem cells can contribute to many or all tissues of aprenatal, postnatal or adult animal. A standard art-accepted test, suchas the ability to form a teratoma in 8-12 week old SCID mice, can beused to establish the pluripotency of a cell population, howeveridentification of various pluripotent stem cell characteristics can alsobe used to detect pluripotent cells. Cell pluripotency is a continuum,ranging from the completely pluripotent cell that can form every cell ofthe embryo proper, e.g., embyronic stem cells and iPSCs, to theincompletely or partially pluripotent cell that can form cells of allthree germ layers but that may not exhibit all the characteristics ofcompletely pluripotent cells, such as, for example, germlinetransmission or the ability to generate a whole organism. In particularembodiments, the pluripotency of a cell is increased from anincompletely or partially pluripotent cell to a more pluripotent cellor, in certain embodiments, a completely pluripotent cell. Pluripotencycan be assessed, for example, by teratoma formation, germ-linetransmission, and tetraploid embryo complementation. In someembodiments, expression of pluripotency genes or pluripotency markers asdiscussed elsewhere herein, can be used to assess the pluripotency of acell.

“Pluripotent stem cell characteristics” refer to characteristics of acell that distinguish pluripotent stem cells from other cells. Theability to give rise to progeny that can undergo differentiation, underthe appropriate conditions, into cell types that collectivelydemonstrate characteristics associated with cell lineages from all ofthe three germinal layers (endoderm, mesoderm, and ectoderm) is apluripotent stem cell characteristic. Expression or non-expression ofcertain combinations of molecular markers are also pluripotent stem cellcharacteristics. For example, human pluripotent stem cells express atleast some, and optionally all, of the markers from the followingnon-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP,Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog. Cell morphologiesassociated with pluripotent stem cells are also pluripotent stem cellcharacteristics.

The term “library” is used according to its common usage in the art, todenote a collection of molecules, optionally organized and/or catalogedin such a way that individual members can be identified. Libraries caninclude, but are not limited to, combinatorial chemical libraries,natural products libraries, and peptide libraries.

A “recombinant” polynucleotide is a polynucleotide that is not in itsnative state, e.g., the polynucleotide comprises a nucleotide sequencenot found in nature, or the polynucleotide is in a context other thanthat in which it is naturally found, e.g., separated from nucleotidesequences with which it typically is in proximity in nature, or adjacent(or contiguous with) nucleotide sequences with which it typically is notin proximity. For example, the sequence at issue can be cloned into avector, or otherwise recombined with one or more additional nucleicacid.

“Expression cassette” refers to a polynucleotide comprising a promoteror other regulatory sequence operably linked to a sequence encoding aprotein.

The terms “promoter” and “expression control sequence” are used hereinto refer to an array of nucleic acid control sequences that directtranscription of a nucleic acid. As used herein, a promoter includesnecessary nucleic acid sequences near the start site of transcription,such as, in the case of a polymerase II type promoter, a TATA element. Apromoter also optionally includes distal enhancer or repressor elements,which can be located as much as several thousand base pairs from thestart site of transcription. Promoters include constitutive andinducible promoters. A “constitutive” promoter is a promoter that isactive under most environmental and developmental conditions. An“inducible” promoter is a promoter that is active under environmental ordevelopmental regulation. The term “operably linked” refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter, or array of transcription factor binding sites) anda second nucleic acid sequence, wherein the expression control sequencedirects transcription of the nucleic acid corresponding to the secondsequence.

A “heterologous sequence” or a “heterologous nucleic acid”, as usedherein, is one that originates from a source foreign to the particularhost cell, or, if from the same source, is modified from its originalform. Thus, a heterologous expression cassette in a cell is anexpression cassette that is not endogenous to the particular host cell,for example by being linked to nucleotide sequences from an expressionvector rather than chromosomal DNA, being linked to a heterologouspromoter, being linked to a reporter gene, etc.

The terms “agent” or “test compound” refer to any compound useful in thescreening assays described herein. An agent can be, for example, anorganic compound, a polypeptide (e.g., a peptide or an antibody), anucleic acid (e.g., DNA, RNA, double-stranded, single-stranded, anoligonucleotide, antisense RNA, small inhibitory RNA, micro RNA, aribozyme, etc.), an oligosaccharide, a lipid. Usually, the agents usedin the present screening methods have a molecular weight of less than10,000 daltons, for example, less than 8000, 6000, 4000, 2000 daltons,e.g., between 50-1500, 500-1500, 200-2000, 500-5000 daltons. The testcompound can be in the form of a library of test compounds, such as acombinatorial or randomized library that provides a sufficient range ofdiversity. Test compounds are optionally linked to a fusion partner,e.g., targeting compounds, rescue compounds, dimerization compounds,stabilizing compounds, addressable compounds, and other functionalmoieties. Conventionally, new chemical entities with useful propertiesare generated by identifying a test compound (called a “lead compound”)with some desirable property or activity, e.g., ability to inducepluripotency under certain conditions such as are described herein,creating variants of the lead compound, and evaluating the property andactivity of those variant compounds. Often, high throughput screening(HTS) methods are employed for such an analysis.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to deoxyribonucleotides or ribonucleotides and polymersthereof in either single- or double-stranded form. The term encompassesnucleic acids containing known nucleotide analogs or modified backboneresidues or linkages, which are synthetic, naturally occurring, andnon-naturally occurring, which have similar binding properties as thereference nucleic acid, and which are metabolized in a manner similar tothe reference nucleotides. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences, as well as thesequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

“Inhibitors,” “activators,” and “modulators” of expression or ofactivity are used to refer to inhibitory, activating, or modulatingmolecules, respectively, identified using in vitro and in vivo assaysfor expression or activity of a described target protein (or encodingpolynucleotide), e.g., ligands, agonists, antagonists, and theirhomologs and mimetics. The term “modulator” includes inhibitors andactivators. Inhibitors are agents that, e.g., inhibit expression or bindto, partially or totally block stimulation or protease inhibitoractivity, decrease, prevent, delay activation, inactivate, desensitize,or down regulate the activity of the described target protein, e.g.,antagonists. Activators are agents that, e.g., induce or activate theexpression of a described target protein or bind to, stimulate,increase, open, activate, facilitate, enhance activation or proteaseinhibitor activity, sensitize or up regulate the activity of describedtarget protein (or encoding polynucleotide), e.g., agonists. Modulatorsinclude naturally occurring and synthetic ligands, antagonists andagonists (e.g., small chemical molecules, antibodies and the like thatfunction as either agonists or antagonists). Such assays for inhibitorsand activators include, e.g., applying putative modulator compounds tocells expressing the described target protein and then determining thefunctional effects on the described target protein activity, asdescribed above. Samples or assays comprising described target proteinthat are treated with a potential activator, inhibitor, or modulator arecompared to control samples without the inhibitor, activator, ormodulator to examine the extent of effect. Control samples (untreatedwith modulators) are assigned a relative activity value of 100%.Inhibition of a described target protein is achieved when the activityvalue relative to the control is about 80%, optionally 50% or 25, 10%,5% or 1%. Activation of the described target protein is achieved whenthe activity value relative to the control is 110%, optionally 150%,optionally 200, 300%, 400%, 500%, or 1000-3000% or more higher.

An “Oct polypeptide” refers to any of the naturally-occurring members ofOctamer family of transcription factors, or variants thereof thatmaintain transcription factor activity, similar (within at least 50%,80%, or 90% activity) compared to the closest related naturallyoccurring family member, or polypeptides comprising at least theDNA-binding domain of the naturally occurring family member, and canfurther comprise a transcriptional activation domain. Exemplary Octpolypeptides include, Oct-1, Oct-2, Oct-3/4, Oct-6, Oct-7, Oct-8, Oct-9,and Oct-11. e.g. Oct3/4 (referred to herein as “Oct4”) contains the POUdomain, a 150 amino acid sequence conserved among Pit-1, Oct-1, Oct-2,and uric-86. See, Ryan, A. K. & Rosenfeld, M. G. Genes Dev. 11,1207-1225 (1997). In some embodiments, variants have at least 85%, 90%,or 95% amino acid sequence identity across their whole sequence comparedto a naturally occurring Oct polypeptide family member such as to thoselisted above or such as listed in Genbank accession number NP_002692.2(human Oct4) or NP_038661.1 (mouse Oct4). Oct polypeptides (e.g.,Oct3/4) can be from human, mouse, rat, bovine, porcine, or otheranimals. Generally, the same species of protein will be used with thespecies of cells being manipulated.

A “Klf polypeptide” refers to any of the naturally-occurring members ofthe family of Krüppel-like factors (Klfs), zinc-finger proteins thatcontain amino acid sequences similar to those of the Drosophilaembryonic pattern regulator Krüppel, or variants of thenaturally-occurring members that maintain transcription factor activitysimilar (within at least 50%, 80%, or 90% activity) compared to theclosest related naturally occurring family member, or polypeptidescomprising at least the DNA-binding domain of the naturally occurringfamily member, and can further comprise a transcriptional activationdomain. See, Dang, D. T., Pevsner, J. & Yang, V. W. Cell Biol. 32,1103-1121 (2000). Exemplary Klf family members include, Klf1, Klf2,Klf3, Klf-4, Klf5, Klf6, K1f7, Klf8, K119, Klf10, Klf11, Klf12, Klf13,Klf14, Klf15, Klf16, and Klf17. Klf2 and Klf-4 were found to be factorscapable of generating iPS cells in mice, and related genes Klf1 and Klf5did as well, although with reduced efficiency. See, Nakagawa, et al.,Nature Biotechnology 26:101-106 (2007). In some embodiments, variantshave at least 85%, 90%, or 95% amino acid sequence identity across theirwhole sequence compared to a naturally occurring Klf polypeptide familymember such as to those listed above or such as listed in Genbankaccession number CAX16088 (mouse Klf4) or CAX14962 (human Klf4). Klfpolypeptides (e.g., Klf1, Klf4, and Klf5) can be from human, mouse, rat,bovine, porcine, or other animals. Generally, the same species ofprotein will be used with the species of cells being manipulated. To theextent a Klf polypeptide is described herein, it can be replaced with anestrogen-related receptor beta (Essrb) polypeptide. Thus, it is intendedthat for each Klf polypeptide embodiment described herein, acorresponding embodiment using Essrb in the place of a Klf4 polypeptideis equally described.

A “Myc polypeptide” refers any of the naturally-occurring members of theMyc family (see, e.g., Adhikary, S. & Eilers, M. Nat. Rev. Mol. CellBiol. 6:635-645 (2005)), or variants thereof that maintain transcriptionfactor activity similar (within at least 50%, 80%, or 90% activity)compared to the closest related naturally occurring family member, orpolypeptides comprising at least the DNA-binding domain of the naturallyoccurring family member, and can further comprise a transcriptionalactivation domain. Exemplary Myc polypeptides include, e.g., c-Myc,N-Myc and L-Myc. In some embodiments, variants have at least 85%, 90%,or 95% amino acid sequence identity across their whole sequence comparedto a naturally occurring Myc polypeptide family member, such as to thoselisted above or such as listed in Genbank accession number CAA25015(human Myc). Myc polypeptides (e.g., c-Myc) can be from human, mouse,rat, bovine, porcine, or other animals. Generally, the same species ofprotein will be used with the species of cells being manipulated.

A “Sox polypeptide” refers to any of the naturally-occurring members ofthe SRY-related HMG-box (Sox) transcription factors, characterized bythe presence of the high-mobility group (HMG) domain, or variantsthereof that maintain transcription factor activity similar (within atleast 50%, 80%, or 90% activity) compared to the closest relatednaturally occurring family member, or polypeptides comprising at leastthe DNA-binding domain of the naturally occurring family member, and canfurther comprise a transcriptional activation domain. See, e.g., Dang,D. T., et al., Int. J. Biochem. Cell Biol. 32:1103-1121 (2000).Exemplary Sox polypeptides include, e.g., Sox1, Sox-2, Sox3, Sox4, Sox5,Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17,Sox18, Sox-21, and Sox30. Sox1 has been shown to yield iPS cells with asimilar efficiency as Sox2, and genes Sox3, Sox15, and Sox18 have alsobeen shown to generate iPS cells, although with somewhat less efficiencythan Sox2. See, Nakagawa, et al., Nature Biotechnology 26:101-106(2007). In some embodiments, variants have at least 85%, 90%, or 95%amino acid sequence identity across their whole sequence compared to anaturally occurring Sox polypeptide family member such as to thoselisted above or such as listed in Genbank accession number CAA83435(human Sox2). Sox polypeptides (e.g., Sox1, Sox2, Sox3, Sox15, or Sox18)can be from human, mouse, rat, bovine, porcine, or other animals.Generally, the same species of protein will be used with the species ofcells being manipulated.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is based in the surprising finding that ALK5inhibitors significantly improve the maintenance, and optionally,induction of pluripotency in cells. Combination of an inhibitor of ALK5with a MAPK inhibitor (e.g., a MEK inhibitor, an Erk inhibitor or a p38inhibitor) or combination of an ALK5 inhibitor with an FGF pathwayinhibitor (e.g., an FGF receptor inhibitor) allows for:

maintenance of pluripotency of cells with new functional properties thatare defined below and are significantly different from the conventionalhuman embryonic stem cells or induced pluripotent stem cells describedpreviously (e.g. in U.S. Pat. Nos. 5,843; 6,200,806; and 7,029,913) andmore similar to mESC characteristics; and

greatly improved efficiency and stability of cells compared to, forexample, methods for maintaining pluripotency known previously (e.g.,involving GSK3 and MEK inhibitors—see, WO2008/015418). Indeed, it issurprising that an inhibitor of ALK5 is effective in improvingmaintenance of pluripotency in part because the art to date has focusedon agonizing, not antagonizing, the TGFβ pathway to stimulatepluripotency. See, e.g., WO 2008/056173.

The invention provides in part for cell cultures comprising an ALK5inhibitor (or other TGFβ/activin pathway inhibitor) and an MAPKinhibitor or a FGF signaling pathway inhibitor, optionally comprising amammalian cell that is already pluripotent or that is to be, or hasbeen, induced to pluripotency in the presence of the inhibitors.Optionally, the cell cultures can also include a GSK3β inhibitor and/orLeukemia Inhibitory Factor (LIF). Other media components and conditionscan be as generally known in the art and can include, e.g., basal mediacomponents, vitamins, minerals, etc.

The ability to maintain cells in pluripotency allows for study and useof such cells in many ways that would otherwise be impossible. Forexample, many pluripotent stems cells quickly differentiate or die inculture and therefore do not allow for screening assays, geneticengineering, and other uses where it is necessary or convenient tomaintain pluripotency for a certain time period or through multiple cellpassages (e.g., cell divisions). The present invention allows for one tocircumvent such problems.

II. Cultures

Cell cultures are provided that include an ALK5 inhibitor (or otherTGFβ/activin pathway inhibitor), optionally with a MAPK (e.g., a MEK orErk or p38 inhibitor) or FGF signaling pathway inhibitor (e.g., an FGFreceptor inhibitor) and, to induce or maintain pluripotency of amammalian cell. In some embodiments, the cells cultured with suchinhibitors have the characteristics described in the examples, includingbut not limited to, forming domed colonies in cultures, expression ofESC markers (e.g., Oct4, Sox2, and Nanog), having nearly completedemethylation of the Oct4 promoter, expressing Rex-1 and ALP (e.g.,markers of ESCs and early Epiblasts that are absent in post-implantationstage Epiblasts and EpiSCs), the ability to differentiate in vitro intoendoderm, neuroectoderm, and mesoderm as well as in vivo pluripotencycharacteristics such as the ability to form teratoma (e.g., in SCIDmice) and for non-human cells, the ability to form chimeric progeny wheninjected into blastocysts and implanted into a receptive uterus.Moreover, in some embodiments, the cells in the cultures retain suchcharacteristics for multiple cell passages, e.g., at least 1, 2, 3, 4,5, 7, 10, 20, 30, or more while in the same culture conditions.

The cell cultures can optionally also include one or both of a GSk3βinhibitor and LIF. As explained in the examples, the presence of LIF canin some embodiments improve long-term maintenance of pluripotent cells(e.g., over more than 10 passages) and thus a sufficient amount of LIFcan be included in the cultures to allow for long-term maintenance ofpluripotency. Further, with or without LIF, a sufficient amount of aGSK3β inhibitor can also be included. In some embodiments, the amount ofthe GSK3β inhibitor is sufficient to improve efficiency of the culture,i.e., the number of positive pluripotent colonies that are formed.

The amount of each inhibitor can vary and be determined for optimumadvantage depending on the precise culture conditions, specificinhibitors used, and type of cell cultured. In some embodiments, thecultures of the invention include 0.05-10 μM, e.g., 0.1-1 μM, e.g., 0.5μM of an ALK5 inhibitor (e.g., A-83-01, and 0.1˜20 μM, e.g., 2˜10 μM ofSB431542). The inventors have found that TGF-β RI Kinase Inhibitor II[2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine] can beused as an ALK5 inhibitor, as described herein, for example at aconcentrations of about 1.5 μM. Thus in some embodiments, cultures ofthe invention include 0.05-20 μM, e.g., 0.1-10 μM of TGF-β RI KinaseInhibitor II. In some embodiments, the cultures of the invention include10 nM-5 μM, e.g., 50 nM-1 μM of an FGF pathway inhibitor (e.g.,PD173074). In some embodiments, the cultures of the invention include0.05-50 μM, e.g., 0.1-5 μM, e.g., 0.5 μM of a MEK inhibitor (e.g.,PD0325901). In some embodiments, the cultures of the invention include0.05-20 μM, e.g., 0.5-5 μM, e.g., 3 μM of a GSK3β inhibitor (e.g.,CHIR99021).

TGF β receptor (e.g., ALK5) inhibitors can include antibodies to,dominant negative variants of and antisense nucleic acids that targetTGF β receptors (e.g., ALK5). Exemplary TGFβ receptor/ALK5 inhibitorsinclude, but are not limited to, SB431542 (see, e.g., Inman, et al.,Molecular Pharmacology 62(1):65-74 (2002)), A-83-01, also known as3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide(see, e.g., Tojo, et al., Cancer Science 96(11):791-800 (2005), andcommercially available from, e.g., Toicris Bioscience); TGF-β RI KinaseInhibitor II[2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine];2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine,Wnt3a/BIO (see, e.g., Dalton, et al., WO2008/094597, herein incorporatedby reference), BMP4 (see, Dalton, supra), GW788388(-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide)(see, e.g., Gellibert, et al., Journal of Medicinal Chemistry49(7):2210-2221 (2006)), SM16 (see, e.g., Suzuki, et al., CancerResearch 67(5):2351-2359 (2007)), IN-1130(3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide)(see, e.g., Kim, et al., Xenobiotica 38(3):325-339 (2008)), GW6604(2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine) (see, e.g., deGouville, et al., Drug News Perspective 19(2):85-90 (2006)), SB-505124(2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride) (see, e.g., DaCosta, et al., Molecular Pharmacology65(3):744-752 (2004)) and pyrimidine derivatives (see, e.g., thoselisted in Stiefl, et al., WO2008/006583, herein incorporated byreference). Without intending to limit the scope of the invention, it isbelieved that ALK5 inhibitors affect the mesenchymal to epithelialconversion/transition (MET) process. TGFβ/activin pathway is a driverfor epithelial to mesenchymal transition (EMT). Therefore, inhibitingthe TGFβ/activin pathway can facilitate MET (i.e. reprogramming)process.

In view of the data herein showing the effect of inhibiting ALK5, it isbelieved that inhibition of the TGFβ/activin pathway will have similareffects. Thus, any inhibitor (e.g., upstream or downstream) of theTGFβ/activin pathway can be used in combination with, or instead of,ALK5 inhibitors as described in each paragraph herein. ExemplaryTGFβ/activin pathway inhibitors include but are not limited to: TGFβreceptor inhibitors, inhibitors of SMAD 2/3 phosphorylation, inhibitorsof the interaction of SMAD 2/3 and SMAD 4, and activators/agonists ofSMAD 6 and SMAD 7. Furthermore, the categorizations described below aremerely for organizational purposes and one of skill in the art wouldknow that compounds can affect one or more points within a pathway, andthus compounds may function in more than one of the defined categories.

TGFβ receptor inhibitors can include antibodies to, dominant negativevariants of and antisense nucleic acids that target TGFβ receptors.Specific examples of inhibitors include but are not limited to SU5416;2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride (SB-505124); lerdelimumb (CAT-152); metelimumab (CAT-192);GC-1008; ID11; AP-12009; AP-11014; LY550410; LY580276; LY364947;LY2109761; SB-505124; SB-431542; SD-208; SM16; NPC-30345; Ki26894;SB-203580; SD-093; Gleevec; 3,5,7,2′,4′-pentahydroxyflavone (Morin);activin-M108A; P144; soluble TBR2-Fc; and antisense transfected tumorcells that target TGFβ receptors. (See, e.g., Wrzesinski, et al.,Clinical Cancer Research 13(18):5262-5270 (2007); Kaminska, et al., ActaBiochimica Polonica 52(2):329-337 (2005); and Chang, et al., Frontiersin Bioscience 12:4393-4401 (2007).)

Inhibitors of SMAD 2/3 phosphorylation can include antibodies to,dominant negative variants of and antisense nucleic acids that targetSMAD2 or SMAD3. Specific examples of inhibitors include PD169316;SB203580; SB-431542; LY364947; A77-01; and3,5,7,2′,4′-pentahydroxyflavone (Morin). (See, e.g., Wrzesinski, supra;Kaminska, supra; Shimanuki, et al., Oncogene 26:3311-3320 (2007); andKataoka, et al., EP1992360, incorporated herein by reference.)

Inhibitors of the interaction of SMAD 2/3 and smad4 can includeantibodies to, dominant negative variants of and antisense nucleic acidsthat target SMAD2, SMAD3 and/or smad4. Specific examples of inhibitorsof the interaction of SMAD 2/3 and SMAD4 include but are not limited toTrx-SARA, Trx-xFoxH1b and Trx-Lef1. (See, e.g., Cui, et al., Oncogene24:3864-3874 (2005) and Zhao, et al., Molecular Biology of the Cell,17:3819-3831 (2006).)

Inhibitors of MEK can include antibodies to, dominant negative variantsof and antisense nucleic acids that target MEK. Specific examples of MEKinhibitors include, but are not limited to, PD0325901, (see, e.g.,Rinehart, et al., Journal of Clinical Oncology 22: 4456-4462 (2004)),PD98059 (available, e.g., from Cell Signaling Technology), U0126(available, for example, from Cell Signaling Technology), SL 327(available, e.g., from Sigma-Aldrich), ARRY-162 (available, e.g., fromArray Biopharma), PD184161 (see, e.g., Klein, et al., Neoplasia 8:1-8(2006)), PD184352 (CI-1040) (see, e.g., Mattingly, et al., The Journalof Pharmacology and Experimental Therapeutics 316:456-465 (2006)),sunitinib (see, e.g., Voss, et al., US2008004287 incorporated herein byreference), sorafenib (see, Voss supra), Vandetanib (see, Voss supra),pazopanib (see, e.g., Voss supra), Axitinib (see, Voss supra) and PTK787(see, Voss supra).

Currently, several MEK inhibitors are undergoing clinical trialevaluations. CI-1040 has been evaluate in Phase I and II clinical trialsfor cancer (see, e.g., Rinehart, et al., Journal of Clinical Oncology22(22):4456-4462 (2004)). Other MEK inhibitors being evaluated inclinical trials include PD184352 (see, e.g., English, et al., Trends inPharmaceutical Sciences 23(1):40-45 (2002)), BAY 43-9006 (see, e.g.,Chow, et al., Cytometry (Communications in Clinical Cytometry) 46:72-78(2001)), PD-325901 (also PD0325901), GSK1120212, ARRY-438162, RDEA119,AZD6244 (also ARRY-142886 or ARRY-886), RO5126766, XL518 and AZD8330(also ARRY-704). (See, e.g., information from the National Institutes ofHealth located on the World Wide Web at clinicaltrials.gov as well asinformation from the Nation Cancer Institute located on the World WideWeb at cancer.gov/clinicaltrials.

p38 (also known as CSBP, mHOG1, RK and SAPK2) inhibitors can includeantibodies to, dominant negative variants of and antisense nucleic acidsthat target p38. Specific examples of inhibitors include but are notlimited to SB203580(4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole);SB202190(4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5(4-pyridyl)-1H-imidazole); SB220025;N-(3-tert-butyl-1-methyl-5-pyrazolyl)-N′-(4-(4-pyridinylmethyl)phenyl)urea;RPR 200765A; UX-745; UX-702; UX-850; SC10-469; RWJ-67657 (RW JohnsonPharmaceutical Research Institute); RDP-58 (SangStat Medical Corp.;acquired by Genzyme Corp.); Scios-323 (SCIO 323; Scios Inc.); Scios-469(SCIO-469; Scios Inc.); MKK3/MKK6 inhibitors (Signal Research Division);p38/MEK modulators (Signal Research Division); SB-210313 analogs;SB-238039; HEP-689 (SB 235699); SB-239063; SB-239065; SB-242235(SmithKline Beecham Pharmaceuticals); VX-702 and VX-745 (VertexPharmaceuticals Inc.); AMG-548 (Amgen Inc.); Astex p38 kinase inhibitors(Astex Technology Ltd.); RPR-200765 analogs (Aventis SA); Bayer p38kinase inhibitors (Bayer Corp.); BIRB-796 (Boehringer IngelheimPharmaceuticals Inc.); Celltech p38 MAP kinase inhibitor (Celltech Groupplc.); FR-167653 (Fujisawa Pharmaceutical Co. Ltd.); SB-681323 andSB-281832 (GlaxoSmithKline plc); LEO Pharmaceuticals MAP kinaseinhibitors (LEO Pharma A/S); Merck Co. p38 MAP kinase inhibitors (Merckresearch Laboratories); SC-040 and SC-XX906 (Monsanto Co.); adenosine A3antagonists (Novartis AG); p38 MAP kinase inhibitors (Novartis PharmaAG); CNI-1493 (Picower Institute for Medical Research); RPR-200765A(Rhone-Poulenc Rorer Ltd.); and Roche p38 MAP kinase inhibitors (e.g.,RO3201195 and RO4402257; Roche Bioscience). See, e.g., Roux, et al.,Microbiology and Molecular Biology Reviews 68(2):320-344 (2004);Engelman, et al., Journal of Biological Chemistry 273(48):32111-32120(1998); Jackson, et al., Journal of Pharmacology and ExperimentalTherapeutics 284(2):687-692 (1998); Kramer, et al., Journal ofBiological Chemistry 271(44):27723-27729 (1996); and Menko, et al.,US20080193504.

Additional inhibitors of p38 include but are not limited to1,5-diaryl-substituted pyrazole and substituted pyrazole compounds (U.S.Pat. No. 6,509,361 and U.S. Pat. No. 6,335,336); substituted pyridylcompounds (US20030139462); quinazoline derivatives (U.S. Pat. No.6,541,477, U.S. Pat. No. 6,184,226, U.S. Pat. No. 6,509,363 and U.S.Pat. No. 6,635,644); aryl ureas and heteroaryl analogues (U.S. Pat. No.6,344,476); heterocyclic ureas (WO1999/32110); other urea compounds(WO1999/32463, WO1998/52558, WO1999/00357 and WO1999/58502); andsubstituted imidazole compounds and substituted triazole compounds (U.S.Pat. No. 6,560,871 and U.S. Pat. No. 6,599,910).

Inhibitors of Erk can include antibodies to, dominant negative variantsof and antisense nucleic acids that target Erk. Specific examples of Erkinhibitors include but are not limited to PD98059 (see, e.g., Zhu, etal., Oncogene 23:4984-4992 (2004)), U0126 (see, Zhu, supra), FR180204(see, e.g., Ohori, Drug News Perspective 21(5):245-250 (2008)),sunitinib (see, e.g., Ma, et al., US2008004287 incorporated herein byreference), sorafenib (see, Ma, supra), Vandetanib (see, Ma, supra),pazopanib (see, Ma, supra), Axitinib (see, Ma, supra) and PTK787 (see,Ma, supra). Erk inhibitors can include molecules that inhibit Erk aloneor that also inhibit a second target as well. For example, in someembodiments, the Erk inhibitor is:

in which:

-   R₁ is selected from hydrogen, C₁₋₆alkyl, C₂-₆alkenyl,    C₆₋₁₀aryl-0-4alkyl, C₅₋₁₀heteroaryl-₀₋₄alkyl,    C₃₋₁₀cycloalkyl-C₀₋₄alkyl and C₃₋₁₀heterocycloalkyl-C₀₋₄alkyl;    wherein any alkyl or alkenyl of R₁ is optionally substituted by one    to three radicals independently selected from halo, hydroxy,    C₁₋₆alkyl and —NR₂R₃; wherein any aryl, heteroaryl, cycloalkyl or    heterocycloalkyl of R₁ is optionally substituted by one to three    radicals selected from halo, hydroxy, cyano, C₁₋₆alkyl, C₁₋₆alkoxy,    C₂-₆alkenyl, halo-substituted-alkyl, halo-substituted-alkoxy,    —XNR₂R₃, —XOXNR₂R₃, —XNR₂S(O)₀₋₂R₃, —XC(O)NR₂R₃, —XNR₂C(O)XOR₂,    —XNR₂C(O)NR₂R₃, —XNR₂XNR₂R₃, —XC(O)NR₂XNR₂R₃, —XNR₂XOR₂, —XOR₂,    —XNR₂C(═NR₂)NR₂R₃, —XS(O)₀₋₂R₄, —XNR₂C(O)R₂, —XNR₂C(O)XNR₂R₃,    —XNR₂C(O)R₄, —XC(O)R₄, —XR₄, —XC(O)OR₃ and —XS(O)₀₋₂NR₂R₃; wherein X    is a bond or C₁₋₄alkylene; R₂ and R₃ are independently selected from    hydrogen, C₁₋₆alkyl and C₃₋₁₂cycloalkyl; and R₄ is    C₃₋₁₀heterocycloallcyl optionally substituted with 1 to 3 radicals    selected from C₁₋₆alkyl, —XNR₂R₃, —XNR₂XNR₂R₂, XNR₂XOR₂ and —XOR₂;    wherein X, R₂ and R₃ are as described above; and the N-oxide    derivatives, prodrug derivatives, protected derivatives, individual    isomers and mixture of isomers thereof; and the pharmaceutically    acceptable salts and solvates (e.g. hydrates) of such compounds or    as otherwise described in WO 06/135824.

Inhibitors of the FGF signaling pathway include, but are not limited toFGF receptor inhibitors. FGF receptor (FGFR) inhibitors can includeantibodies to, dominant negative variants of and antisense nucleic acidsthat target FGFR. Specific examples of FGFR inhibitors include, but arenot limited to, SU6668 (see, e.g., Klenke, BMC Cancer 7:49 (2007)),SU5402(3-[3-(2-Carboxyethyl)-4-methylpyrrol-2-methylidenyl]-2-indolinone) andPD173074 (see, e.g., Bansal, et al., J. Neuro. Res. 74(4):486-493(2003)).

Inhibitors of GSK3 can include antibodies to, dominant negative variantsof and antisense nucleic acids that target GSK3. Specific examples ofGSK3 inhibitors include, but are not limited to, CHIR99021, CHIR98014,AR-A014418 (see, e.g., Gould, et al., The International Journal ofNeuropsychopharmacology 7:387-390 (2004)), CT 99021 (see, e.g., Wagman,Current Pharmaceutical Design 10:1105-1137 (2004)), CT 20026 (see,Wagman, supra), SB216763 (see, e.g., Martin, et al., Nature Immunology6:777-784 (2005)), AR-A014418 (see, e.g., Noble, et al., PNAS102:6990-6995 (2005)), lithium (see, e.g., Gould, et al.,Pharmacological Research 48: 49-53 (2003)), SB 415286 (see, e.g., Frame,et al., Biochemical Journal 359:1-16 (2001)) and TDZD-8 (see, e.g.,Chin, et al., Molecular Brain Research, 137(1-2):193-201 (2005)).Further exemplary GSK3 inhibitors available from Calbiochem (see, e.g.,Dalton, et al., WO2008/094597, herein incorporated by reference),include but are not limited to BIO (2′Z,3′£)-6-Bromoindirubin-3′-oxime(GSK3 Inhibitor IX); BIO-Acetoxime(2′Z,3′E)-6-Bromoindirubin-3′-acetoxime (GSK3 Inhibitor X);(5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine(GSK3-Inhibitor XIII); Pyridocarbazole-cyclopenadienylruthenium complex(GSK3 Inhibitor XV); TDZD-84-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK3beta InhibitorI); 2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3betaInhibitor II); OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione(GSK3beta Inhibitor III); alpha-4-Dibromoacetophenone (GSK3betaInhibitor VII); AR-AO 14418N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3betaInhibitor VIII);3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione(GSK-3beta Inhibitor XI); TWSl 19 pyrrolopyrimidine compound (GSK3betaInhibitor XII); L803 H-KEAPPAPPQSpP-NH2 (SEQ ID NO:1) or itsMyristoylated form (GSK3beta Inhibitor XIII);2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3beta Inhibitor VI);AR-AO144-18; SB216763; and SB415286. Residues of GSK3b that interactwith inhibitors have been identified. See, e.g., Bertrand et al., J. MolBiol. 333(2): 393-407 (2003).

Where inhibitors of a particular gene product are described herein, itshould be understood that the inhibitor can be replaced with an siRNAtargeting the gene encoding the gene product. For example, the presentinvention provides for use of an siRNA that inhibits expression of ALK5in place of an ALK5 inhibitor. Similarly, MEK inhibitors, Erkinhibitors, p38 inhibitors, FGF receptor inhibitors and GSK3β inhibitorscan be replaced with a MEK siRNA, Erk siRNA, p38 siRNA, FGF receptorsiRNA and GSK3β siRNA, respectively. Further, an inhibitory antibody(e.g., a humanized or chimeric antibody) can be used as an inhibitor ofALK5, MEK, Erk, p38, FGF receptor, and GSK3β.

In some embodiments, cells are initially cultured with an epigeneticmodifier followed by a culturing step lacking the modifier but includingthe inhibitors described herein (e.g., and AKL5 inhibitor, MEKinhibitors, Erk inhibitors, p38 inhibitors, FGF receptor inhibitors andGSK3β inhibitors, Leukemia inhibiting Factor (LIF), etc.). Exemplaryepigenetic modulators include an inhibitor of histone H3K4 demethylationor an activator of H3K4 methylation. Exemplary epigenetic modifiersinclude, e.g., histone demethylase inhibitors such as LSD1 inhibitors(e.g., parnate) or MAO inhibitors.

The terms “histone deacetylase inhibitor,” “inhibitor of histonedeacetylase” and “HDAC inhibitor” refer to a compound capable ofinteracting with a histone deacetylase and inhibiting its enzymaticactivity. “Inhibiting histone deacetylase enzymatic activity” meansreducing the ability of a histone deacetylase to remove an acetyl groupfrom a histone. In some embodiments, such reduction of histonedeacetylase activity is at least about50%, more preferably at leastabout 75%, and still more preferably at least about 90%. In otherpreferred embodiments, histone deacetylase activity is reduced by atleast 95% and more preferably by at least 99%.

Some representative HDAC inhibitors include butyric acid, MS-27-275,SAHA, Trichostatin A, apicidin, oxanflatin, FK228, and trapoxin. Theseinhibitors can be divided into several classes based on theirstructures, including short-chain fatty acids (butyrates and Valproicacid), hydroxamic acids (Trichostatin A and SAHA), cyclic tetrapeptides(depsipeptide), benzamides (MS-27-275), and epoxide-containing agents(trapoxin). Most of them inhibit HDACs in a reversible manner excepttrapoxin, which possesses an epoxide group capable of irreversiblyalkylating HDACs. The reversible inhibitors generally have a longaliphatic tail containing a nucleophilic end, such as —SH or —OH, whichinteracts with the active zinc center located on the bottom of HDACbinding pocket. Other HDAC inhibitors are emerging based on modificationof the listed structures. Most of the new agents are derivatives ofhydroxamic acids, including amide analogues of Trichostatin A (TSA) andthio/phosphorus-based SAHA. Replacement of the amide linkage inMS-27-275 structure with a sulfonamide led to discovery of a new classof potent HDAC inhibitors. Promising HDAC inhibitors that have enteredclinical trials include hydroxamic acid derivative LAQ824, butyric acidderivative Titan, valproic acid, MS-27-275, SAHA, and depsipeptideFK228.

Histone demethylase inhibitors include inhibitors to lysine-specificdemethylase I (LSD1; also known as lysine-specific histone demethylase,BHCl 10 and KIAA0601). International Patent Application No. WO2006/071608 is directed to a method for monitoring eukaryotic histonedemethylase activity, methods for up-regulating and down-regulatingmethylated histone-activated genes, and a method for treating orpreventing a disease (e.g., a hyperproliferative disease such as cancer)by modulating the level of protein or the activity of a histonedemethylase. In view of the importance of gene regulation, and theability to affect gene regulation by inhibiting or modulating LSD1,inhibitors of the enzyme may have significant therapeutic potential; Bi,X. et al., Bioorg. Med. Chem. Lett. 16:3229-3232 (2006) andInternational Patent Application Nos. WO2007/021839 and WO2008/127734describe certain compounds useful as inhibitors of LSD1.

The present invention also provides for a culture medium for maintainingpluripotency of cells. The cell culture media optionally does notinclude a cell. The culture medium can comprise the culture mediacontents described above, albeit with the cells. Such media is usefulculturing cells as described herein.

As provided elsewhere herein, the cells in the cultures can be selectedfrom embryonic stem cells (e.g., human embryonic stem cells (hESCs),primate embryonic stem cells, rat embryonic stem cells, or embryonicstem cells from other animals, optionally non-mouse embryonic stemcells). Alternatively, the cells include induced pluripotent stem cells(iPSCs). In some embodiments, the iPSCs are from humans, primates, rat,or mice, or other non-mouse animals. The iPSCs can be generated fromnon-pluripotent cells as recently as within the previous cell divisionor alternatively, the cells can have been maintained for 1, 2, 3, 4, 5,7, 10, 20, or more cell divisions or passages previously as iPSCs. Inother words, the cell cultures can contain iPSCs that were createdpreviously (e.g., a week, a month, or more previously). One benefit ofthe small molecule combinations provided herein is that in addition andseparate to their use in reprogramming, they allow one to maintain adesired pluripotency for what appears to be an indefinite period oftime.

The present invention provides for pluripotent cells in a mixture withone or more inhibitor as described herein. In some embodiments, thecompound is in the mixture at a concentration sufficient to induce orimprove efficiency of induction to pluripotency. For example, in someembodiments, the compounds are in a concentration of at least 0.1 nM,e.g., at least 1, 10, 100, 1000, 10000, or 100000 nM, e.g., between 0.1nM and 100000 nM, e.g., between 1 nM and 10000 nM, e.g., between 10 nMand 10000 nM, between 0.01 μM and 5 μM, between 0.1 μM and 5 μM. Forexample, A-83-01 can be used in a concentration of about 0.1 μM to about0.5 μM, e.g., 0.25 μM to about 0.5 μM. In some embodiments, theconcentration of A-83-01 is 0.25 μM. In some embodiments, theconcentration of A-83-01 is 0.5 μM. CHIR99021 can be used in aconcentration of about 3 μM. PD325901 can be used in a concentration ofabout 0.5 μM. PD173074 can be used in a concentration of about 0.1 μM.In some embodiments, the mixtures are in a synthetic vessel (e.g., atest tube, Petri dish, etc.). Thus, in some embodiments, the cells areisolated cells (not part of an animal). In some embodiments, the cellsare isolated from an animal (human or non-human), placed into a vessel,contacted with one or more compound as described herein. The cells canbe subsequently cultured and optionally, inserted back into the same ora different animal, optionally after the cells have been stimulated tobecome a particular cell type or lineage.

In some embodiments, the cells comprise an expression cassette forheterologous expression of at least one or more of an Oct polypeptide, aMyc polypeptide, a Sox polypeptide and a Klf polypeptide. In someembodiments, the cells do not include an expression cassette to expressany of the Oct, Myc, Sox of Klf polypeptides. Cells with or without suchexpression cassettes are useful, for example, screening methods asdescribed herein.

III. Cells

Cells can be pluripotent prior to initial contact with the inhibitors ofthe invention or the cells can be contacted with one or more of theinhibitors of the invention and then induced to pluripotency (e.g., byintroduction of the appropriate transcription factors and/or by contactwith the appropriate small molecules to induce pluripotency).

Any animal cells can be used in the methods of the invention. Thus, forexample, in some embodiments, the cells are mammalian cells. Exemplarymammalian cells include, but are not limited to, human cells ornon-human cells, including but not limited to rat, mouse (e.g., SCID orother mice), pig, bovine, ovine, canine, feline, and primate (e.g.,rhesus monkey, chimpanzee, etc.).

Pluripotent cells used according to the methods of the invention can beeither naturally-occurring stem cells or can be induced pluripotentcells. Exemplary naturally-occurring stem cells include, e.g., embryonicstem cells. Methods of isolating embryonic stems cells are well known.See, e.g., Matsui et al., Cell 70:841, 1992; Thomson et al., U.S. Pat.No. 5,843,780; Thomson et al., Science 282:114, 1998; Shamblott et al.,Proc. Natl. Acad. Sci. USA 95:13726, 1998; Shamblott et al., U.S. Pat.No. 6,090,622; Reubinoff et al., Nat. Biotech. 18:399, 2000; PCTWO00/27995, Iannaccone et al., Dev. Biol. 163:288, 1994; Loring et al.,PCT WO99/27076, Pain et al., Development 122:2339, 1996; U.S. Pat. No.5,340,740; U.S. Pat. No. 5,656,479, Wheeler et al., Reprod. Fertil. Dev.6:563, 1994; Shim et al., Biol. Reprod. 57:1089, 1997. In someembodiments, the stem cells are derived from a blastocyst (e.g.,obtained from a blastocyst) that are subsequently cultured in thepresence of at least an ALK5 inhibitor and an Erk or MEK inhibitor,optionally with other inhibitors as described herein.

A number of ways have now been reported for inducing pluripotency incells. Pluripotency can be induced, for example, by introduction oftranscription factors or otherwise induce or mimic expression of certaintranscription factors. In some embodiments, one or more of the followingtranscription factors are expressed endogenously or recombinantly (e.g.,by introduction of heterologous expression cassettes expressing one ormore transcription factors). Exemplary technologies for induction ofpluripotency include, but are not limited to introduction of at leastone or more expression cassette for expression of at least one ofOct3/4, Sox2, c-Myc, and Klf4 (see, e.g., Takahashi, Cell 131(5):861-872(2007); Cell Stem Cell 2, 10-12 (2008)), optionally with one or moresmall molecules, including but not limited to, an agent that inhibitsH3K9 methylation, e.g., a G9a histone methyltransferase such asBIX01294. See, e.g., Kubicek, et al., Mol. Cell 473-481 (2007).

The pluripotent cells of the invention can be characterized by severalcriteria. In addition to the gene expression, methylation, and in vitroand in vivo characteristics described herein, the pluripotent cells ofthe invention will maintain pluripotency over at least one (e.g., 1, 2,3, 4, 5, 10, 20, etc.) cell divisions in the presence of leukemiainhibitory factor (LIF) and bone morphogenic protein (BMP) or,alternatively, under inhibition of the TGFβ and activin signalingpathway, inhibition of the MAPK signaling pathway, and optionallyinhibition of the FGF pathway. For example, as described herein, animalcells (e.g., human and rat cells) contacted with an ALK5 inhibitor andMEK inhibitor were maintained in pluripotency through multipledivisions. Further, as described herein, inhibition of the TGFβ andactivin signaling pathway (e.g., TGFβ signaling) in conjunction withinhibition of MEK, FGFR and GSK3 has strong reprogramming activity andcan promote partial conversion of EpiSCs to a mESC-like state. Incontrast, conventional hESCs, epiblast stem cells (EpiSCs) and humaninduced pluripotent cells cultured under conventional conditionsdifferentiate when contacted with an inhibitor of ALK5. See, e.g., Saha,et al., Biophys. J. 94: 4123-4133 (2008). It has been reported thatconventional hESCs, EpiSCs and human induced pluripotent cells culturedunder conventional conditions appear dependent on MAPK, FGF, andTGFβ/Activin/Nodal pathway activity for self-renewal, and differentiaterapidly when treated with MEK, FGFR and/or ALK4/5/7 inhibitors (Brons etal., Nature 448, 191-195, 2007; Li et al., Differentiation 75, 299-307,2007; Peerani et al., EMBO J 26, 4744-4755, 2007; Tesar et al., Nature448, 196-199, 2007). In addition, the inventors have found that thecells of the invention (e.g., human or rat cells cultured as describedin the examples) maintain pluripotency (i.e., do not differentiate) inthe presence of an inhibitor of the FGF signaling pathway (e.g.,PD173074). In contrast, hESCs, EpiSCs and human induced pluripotentcells cultured under conventional conditions differentiate whencontacted with PD173074. Notably, mESCs do not differentiate whencontacted with PD173074. Thus, the cells of the present invention (e.g.,cultured as described herein) are in a state more similar to mESCs thanconventionally-cultured hESCs, EpiSCs and human induced pluripotentcells. Similar to mESCs, the cells of the present invention have morecompact and domed colony morphology, while conventionally-culturedhESCs, EpiSCs and human induced pluripotent cells have flat colonymorphology. Like mESCs, the cells of the present invention have theability to give rise to all cell types in vitro, and contribute to anentire animal in vivo, including germline, when placed back intoblastocyts. In contrast, conventionally-cultured hESCs, EpiSCs and humaninduced pluripotent cells are incapable of incorporating into the innercell mass (ICM) and contributing to chimerism. It will be appreciatedthat other animal cells aside from rat and human can be generated withsimilar characteristics using the methods described herein. Exemplaryadditional animal cells include e.g., dogs, cats, pigs, cows, sheep,goats, monkeys and chimpanzees.

Certain markers are helpful to distinguish the cells of the presentinvention from conventionally-cultured hESCs, EpiSCs and human inducedpluripotent cells. For example, Stra8, Dppa3, Gbx2, Pecam1, and Klf4express at a higher level in the human cells of the present invention ascompared to their expression levels in conventionally-cultured hESCs,epiSCs and human induced pluripotent cells. In contrast, many lineagespecific genes, e.g., Foxa2, Otx2, Lefty1, Gata6, Sox17, Cer1, expressat a higher level in epiSCs and conventional human ES cells, as comparedto their expression levels in the cells of the present invention.Conventionally-cultured human iPSC or ESC cells differentiate whentreated with MEK, FGFR and/or ALK4/5/7 inhibitors (Brons et al., Nature448, 191-195, 2007; Li et al., Differentiation 75, 299-307, 2007;Peerani et al., EMBO J 26, 4744-4755, 2007; Tesar et al., Nature 448,196-199, 2007).

In particular, Gbx2, Dppa3 and Klf4 are useful marker to characterizethe pluripotent animal cells of the invention. These markers are highlyexpressed in the pluripotent cells of the invention. For example, insome embodiments, the cells of the present invention express thesemarkers at a level that is at least 2-fold of their level inconventionally-cultured hESCs, EpiSCs and human induced pluripotentcells, e.g., Hues9 cells (hES facility, Harvard University,http://mcb.harvard.edu/melton/hues/). In some embodiments, theexpression levels of these markers in the pluripotent cells of theinvention are at least 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold orhigher. Other useful markers include an ICM marker Rex1.

In some embodiments, Gbx2 is expressed in the pluripotent cells of theinvention at a level that is at least 5-fold of that in Hues9 cells. Insome embodiments, Klf4 is expressed in the pluripotent cells of theinvention at a level that is at least 2.5-fold of that in Hues9 cells.In some embodiments, Dppa3 is expressed in the pluripotent cells of theinvention at a level that is at least 2-fold of that in Hues9 cells.

Another useful marker for the pluripotent cells of the invention isE-cadherin. Without intending to limit the scope of the invention it isbelieved that E-cadherin plays a role in the pluripotency of the cellsof the invention. In some embodiments, E-cadherin is expressed in thepluripotent cells of the invention at a level that is 2-fold of that inHues9 cells.

Other typical pluripotent markers can be used for characterization ofthe cells of the invention, e.g., Oct4, Sox2, Nanog, SSEA-1, SSEA-3,SSEA4, TRA-1-61, TRA-1-81, and alkaline phosphatase (ALP). These typicalpluripotent markers, or a subset of them, can be useful fordistinguishing the pluripotent cells of the invention fromconventionally-cultured hESCs, EpiSCs and human induced pluripotentcells.

Methods known in the art can be used to characterize the cells of theinvention. Gene expression levels can be detected by, e.g., real-timePCR or real-time RT-PCR (e.g., to detect mRNA), and/or by western blotor other protein detection technique.

The pluripotent cells of the invention differentiate toward mesodermlineages in response to BMP treatment. Conventionally-cultured hESCs,EpiSCs and human induced pluripotent cells, in contrast, generatetrophoblasts or primitive endoderm cells (Brons et al., Nature 448,191-195, 2007; D'Amour et al., Nat Biotechnol 23, 1534-1541, 2005; Xu etal., Nat Biotechnol 20, 1261-1264, 2002).

IV. Transformation

Where transformed cells are desired (e.g., to generate iPSCs or toexpress a desired protein or nucleic acid), the cells contacted with theinhibitors of the invention (e.g., ALK5 inhibitors, MAPK inhibitors, FGFpathway inhibitors, and optionally GSK3β inhibitors) can be transformedbefore the contacting and/or can be transformed following contacting.For example, one advantage of the present invention is that it allowsfor maintenance of pluripotent cells through multiple cell passage andtherefore allows for manipulations and subsequent selection of progenywhile maintaining the pluripotent characteristics of the cells. This isuseful, inter alia, for generation of transgenic animals, includinggeneration of knockout animals via sequence-specific recombinationevents as described herein.

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

In some embodiments, where positive expression of a protein is desired,the species of cell and protein to be expressed is the same. Forexample, if a mouse cell is used, a mouse ortholog is introduced intothe cell. If a human cell is used, a human ortholog is introduced intothe cell.

It will be appreciated that where two or more proteins are to beexpressed in a cell, one or multiple expression cassettes can be used.For example, where one expression cassette is to express multiplepolypeptides, a polycistronic expression cassette can be used.

A. Plasmid Vectors

In certain embodiments, a plasmid vector is contemplated for use totransform a host cell. In general, plasmid vectors containing repliconand control sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector can carrya replication site, as well as marking sequences which are capable ofproviding phenotypic selection in transformed cells.

B. Viral Vectors

The ability of certain viruses to infect cells or enter cells viareceptor-mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). Non-limiting examples of virus vectors that may beused to deliver a nucleic acid of the present invention are describedbelow.

i. Adenoviral Vectors

A particular method for delivery of the nucleic acid involves the use ofan adenovirus expression vector. Although adenovirus vectors are knownto have a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a tissue orcell-specific construct that has been cloned therein. Knowledge of thegenetic organization or adenovirus, a ˜36 kb, linear, double-strandedDNA virus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus et al., Seminar in Virology,200(2):535-546, 1992)).

ii. AAV Vectors

The nucleic acid may be introduced into the cell using adenovirusassisted transfection. Increased transfection efficiencies have beenreported in cell systems using adenovirus coupled systems (Kelleher andVos, Biotechniques, 17(6):1110-7, 1994; Cotten et al., Proc Natl AcadSci USA, 89(13):6094-6098, 1992; Curiel, Nat Immun, 13(2-3):141-64,1994.). Adeno-associated virus (AAV) is an attractive vector system asit has a high frequency of integration and it can infect non-dividingcells, thus making it useful for delivery of genes into mammalian cells,for example, in tissue culture (Muzyczka, Curr Top Microbiol Immunol,158:97-129, 1992) or in vivo. Details concerning the generation and useof rAAV vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797,368,each incorporated herein by reference.

iii. Retroviral Vectors

Retroviruses have promise as gene delivery vectors due to their abilityto integrate their genes into the host genome, transferring a largeamount of foreign genetic material, infecting a broad spectrum ofspecies and cell types and of being packaged in special cell-lines(Miller et al., Am. J. Clin. Oncol., 15(3):216-221, 1992).

In order to construct a retroviral vector, a nucleic acid (e.g., oneencoding gene of interest) is inserted into the viral genome in theplace of certain viral sequences to produce a virus that isreplication-defective. To produce virions, a packaging cell linecontaining the gag, pol, and env genes but without the LTR and packagingcomponents is constructed (Mann et al., Cell, 33:153-159, 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into a special cell line (e.g., bycalcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubinstein, In: Vectors: A survey of molecular cloning vectors andtheir uses, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp.494-513, 1988; Temin, In: Gene Transfer, Kucherlapati (ed.), New York:Plenum Press, pp. 149-188, 1986; Mann et al., Cell, 33:153-159, 1983).The media containing the recombinant retroviruses is then collected,optionally concentrated, and used for gene transfer. Retroviral vectorsare able to infect a broad variety of cell types. However, integrationand stable expression typically involves the division of host cells(Paskind et al., Virology, 67:242-248, 1975).

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, Naldini et al., Science, 272(5259):263-267, 1996;Zufferey et al., Nat Biotechnol, 15(9):871-875, 1997; Blomer et al., JVirol., 71(9):6641-6649, 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136).Some examples of lentivirus include the Human Immunodeficiency Viruses:HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviralvectors have been generated by multiply attenuating the HIV virulencegenes, for example, the genes env, vif, vpr, vpu and nef are deletedmaking the vector biologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat is describedin U.S. Pat. No. 5,994,136, incorporated herein by reference. One maytarget the recombinant virus by linkage of the envelope protein with anantibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

iv. Delivery using Modified Viruses

A nucleic acid to be delivered may be housed within an infective virusthat has been engineered to express a specific binding ligand. The virusparticle will thus bind specifically to the cognate receptors of thetarget cell and deliver the contents to the cell. A novel approachdesigned to allow specific targeting of retrovirus vectors was developedbased on the chemical modification of a retrovirus by the chemicaladdition of lactose residues to the viral envelope. This modificationcan permit the specific infection of hepatocytes via sialoglycoproteinreceptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,Proc. Nat'l Acad. Sci. USA, 86:9079-9083, 1989). Using antibodiesagainst major histocompatibility complex class I and class II antigens,they demonstrated the infection of a variety of human cells that borethose surface antigens with an ecotropic virus in vitro (Roux et al.,1989).

C. Vector Delivery and Cell Transformation

Suitable methods for nucleic acid delivery for transformation of a cell,a tissue or an organism for use with the current invention are believedto include virtually any method by which a nucleic acid (e.g., DNA) canbe introduced into a cell, a tissue or an organism, as described hereinor as would be known to one of ordinary skill in the art. Such methodsinclude, but are not limited to, direct delivery of DNA such as by exvivo transfection (Wilson et al., Science, 244:1344-1346, 1989, Nabeland Baltimore, Nature 326:711-713, 1987), optionally with Fugene6(Roche) or Lipofectamine (Invitrogen), by injection (U.S. Pat. Nos.5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932,5,656,610, 5,589,466 and 5,580,859, each incorporated herein byreference), including microinjection (Harland and Weintraub, J. CellBiol., 101:1094-1099, 1985; U.S. Pat. No. 5,789,215, incorporated hereinby reference); by electroporation (U.S. Pat. No. 5,384,253, incorporatedherein by reference; Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986;Potter et al., Proc. Nat'l Acad. Sci. USA, 81:7161-7165, 1984); bycalcium phosphate precipitation (Graham and Van Der Eb, Virology,52:456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752,1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990); by usingDEAE-dextran followed by polyethylene glycol (Gopal, Mol. Cell Biol.,5:1188-1190, 1985); by direct sonic loading (Fechheimer et al., Proc.Nat'l Acad. Sci. USA, 84:8463-8467, 1987); by liposome mediatedtransfection (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190,1982; Fraley et al., Proc. Nat'l Acad. Sci. USA, 76:3348-3352, 1979;Nicolau et al., Methods Enzymol., 149:157-176, 1987; Wong et al., Gene,10:87-94, 1980; Kaneda et al., Science, 243:375-378, 1989; Kato et al.,J Biol. Chem., 266:3361-3364, 1991) and receptor-mediated transfection(Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem.,262:4429-4432, 1987); each incorporated herein by reference); and anycombination of such methods.

V. Culturing of Cells

Pluripotent cells, including embryonic stem cells and cells induced, orto be induced, to pluripotency can be cultured according to any methodknown in the art. As described herein, in some embodiments, pluripotentcells are cultured with an ALK5 inhibitor and one of a MEK or Erkinhibitor, optionally with a GSK3β inhibitor and/or LIF. Culture mediacan include any other component of culture media as known in the art. Insome embodiments, the culture media include basal media components forcell survival (e.g., vitamins and minerals, optionally in an isotoniccondition). An exemplary basal media is DMEM or a variation thereof,such as DMEM Knockout. The culture can further be supplemented withKnock-out serum replacement (KSP). The cultures of the invention caninclude one or more carbon sources. In some embodiments, the culturescomprise L-analyl-L-glutamine. The cultures can include serum or can beserum-free.

In some embodiments, the cells are cultured in contact with feedercells. Exemplary feeder cells include, but are not limited to fibroblastcells, e.g., mouse embryonic fibroblast (MEF) cells, or x-rayinactivated CF1 feeder cells. Methods of culturing cells on feeder cellsis known in the art.

In some embodiments, the cells are cultured in the absence of feedercells. Cells, for example, can be attached directly to a solid culturesurface (e.g., a culture plate), e.g., via a molecular tether. Theinventors have found that culturing cells induced to pluripotency have amuch greater efficiency of induction to pluripotency (i.e., a greaterportion of cells achieve pluripotency) when the cells are attacheddirectly to the solid culturing surface compared the efficiency ofotherwise identically-treated cells that are cultured on feeder cells.Exemplary molecular tethers include, but are not limited to, matrigel,an extracellular matrix (ECM), ECM analogs, laminin, fibronectin, orcollagen. Those of skill in the art however will recognize that this isa non-limiting list and that other molecules can be used to attach cellsto a solid surface. Methods for initial attachment of the tethers to thesolid surface are known in the art.

VI. Uses for Pluripotent Cells

The present invention allows for the further study and development ofstem cell technologies, including but not limited to, prophylactic ortherapeutic uses. For example, in some embodiments, cells of theinvention (either pluripotent cells cultured in the inhibitors of theinvention or cells derived from such cells and induced to differentiatealong a desired cell fate) are introduced into individuals in needthereof, including but not limited to, individuals in need ofregeneration of an organ, tissue, or cell type. In some embodiments, thecells are originally obtained in a biopsy from an individual; inducedinto pluripotency as described herein, optionally induced todifferentiate (for examples into a particular desired progenitor cell)and then transplanted back into the individual. In some embodiments, thecells are genetically modified prior to their introduction into theindividual.

In some embodiments, the pluripotent cells generated according to themethods of the invention are subsequently induced to form, for example,hematopoietic (stem/progenitor) cells, neural (stem/progenitor) cells(and optionally, more differentiated cells, such as subtype specificneurons, oligodendrocytes, etc), pancreatic cells (e.g., endocrineprogenitor cell or pancreatic hormone-expressing cells), hepatocytes,cardiovascular (stem/progenitor) cells (e.g., cardiomyocytes,endothelial cells, smooth muscle cells), retinal cells, etc.

A variety of methods are known for inducing differentiation ofpluripotent stem cells into desired cell types. A non-limiting list ofrecent patent publications describing methods for inducingdifferentiation of stem cells into various cell fates follows: U.S.Patent Publication No. 2007/0281355; 2007/0269412; 2007/0264709;2007/0259423; 2007/0254359; 2007/0196919; 2007/0172946; 2007/0141703;2007/0134215.

A variety of diseases may be ameliorated by introduction, and optionallytargeting, of pluripotent cells of the invention to a particular injuredtissue or tissue other tissue where pluripotent cells will generate abenefit. Examples of disease resulting from tissue injury include, butare not limited to, neurodegeneration disease, cerebral infarction,obstructive vascular disease, myocardial infarction, cardiac failure,chronic obstructive lung disease, pulmonary emphysema, bronchitis,interstitial pulmonary disease, asthma, hepatitis B (liver damage),hepatitis C (liver damage), alcoholic hepatitis (liver damage), hepaticcirrhosis (liver damage), hepatic insufficiency (liver damage),pancreatitis, diabetes mellitus, Crohn disease, inflammatory colitis,IgA glomerulonephritis, glomerulonephritis, renal insufficiency,decubitus, burn, sutural wound, laceration, incised wound, bite wound,dermatitis, cicatricial keloid, keloid, diabetic ulcer, arterial ulcerand venous ulcer.

In some embodiments, transgenic animals (e.g., non-human animals) aregenerated from pluripotent cells incubated with the inhibitors of theinvention. Such cells can be transgenic cells, knockout lines (e.g.,comprising one or more gene knockout introducing a selectable marker viasite-specific recombination). Pluripotent cells incubated with theinhibitors as described herein can be introduced into a blastocyst froma compatible animal and subsequently introduced into a receptive uterusof an animal and resulting progeny can be selected for cells derivedfrom the pluripotent cells (e.g., via a selectable marker or otherphenotypic characteristics such as fur color). Chimeric progeny can beidentified and lines can be established that pass the characteristics(e.g., a transgene) from the pluripotent cells to progeny. Homozygousanimal lines can be established by breeding sibling animals.

The invention provides for generation of any type of transgenic animalaccording to the present methods. Exemplary animals include non-humanmammals, and non-human primates. Exemplary animals include, e.g., mice(including SCID mice), rats, dogs, cats, pigs, cows, sheep, goats,monkeys and chimpanzees.

Culturing cells with the inhibitors of the invention, and thusmaintaining pluripotency of the cells, conveniently allows for screeningfor cell phenotypes, drug responses, and also allows for screening oflibraries of compounds or other agents (e.g., protein, nucleic acid, orantibodies) for the ability to modulate a pluripotent cell's phenotypeor to induce a desired cellular response. Library screens can bedesigned to screen for a library member's ability to affect essentiallyany desired phenotype. Exemplary phenotypes can include, for example,cellular differentiation, apoptosis or other cell death, cell survival,death, or other phenotype in the presence of an additional compound ordrug of interest, etc.

The agents in the library can be any small chemical compound, or abiological entity, such as a protein, sugar, nucleic acid or lipid.Typically, test agents will be small chemical molecules and peptides.Essentially any chemical compound can be used as a potential agent inthe assays of the invention, although most often compounds that can bedissolved in aqueous or organic (especially DMSO-based) solutions areused. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs, Switzerland) and the like.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

EXAMPLES

SUMMARY: Here, we report successful establishment of novel rat iPSCs(riPSCs), which can be homogenously maintained by LIF and a cocktail ofALK5 inhibitor, GSK3 inhibitor and MEK inhibitor. riPSCs share mouse ESCcharacteristics and most importantly can contribute extensively tochimaeras. We also generated novel human iPSCs (hiPSCs) with “mouseESC-like” characteristics, which can be surprisingly maintained inculture in the presence of MEK inhibitor and ALK5 inhibitor. We proposethat our experiments will provide a framework to generate pluripotentstem cells by reprogramming from rats or other species, whose authenticembryonic stem cells are still not available.

Example 1

This example demonstrates generation and maintenance of rat pluripotentcells.

Generation of novel riPSCs from WB-F344 cells by Oct4/Klf4/Sox2 viraltransduction and chemical cocktails. WB-F344, a diploid rat liverprogenitor cell line (Grisham et al., Proc Soc Exp Biol Med 204, 270-279(1993)), were transduced with Oct4, Sox2 and Klf4 by retroviruses andthen split onto MEF feeder cells in the conventional mESC medium.Compact and alkaline phosphatase (ALP) positive ESC-like colonies wereobserved 10 days after transduction (the efficiency is about 0.4%) (FIG.1A). When the ESC-like colonies were picked up and sub-cultured in thesame medium, however, they quickly differentiated and lost ESCmorphology (FIG. 1B), suggesting that the conventional mESC mediumcondition is not sufficient to maintain the pluripotency of riPSCs.Given the notion that small molecules can inhibit keydifferentiation-inducing pathways, we and others have previouslyidentified and used small molecules to support mESC self-renewal in amore robust manner (Schugar et al., Gene Ther 15, 126-135 (2008); Xu etal., Nat Biotechnol 20, 1261-1264 (2002)). Based on such chemicalstrategy and the signaling differences for maintaining self-renewal ofmESC and EpiSC/hESC, we selected MEK inhibitor PD0325901, ALK5 (theprinciple type I receptor of TGF β signaling) inhibitor A-83-01, GSK3βinhibitor CHIR99021, and FGFR inhibitor PD173074, and tested the effectsof different small molecule combination on maintaining the pluripotencyof riPSCs. Using combination of 0.5 μM PD0325901 and 3 μM CHIR99021,riPSCs can be short-term maintained in culture but showing extensivespontaneous differentiation (FIG. 1C). After serial passages, cellsgrown under this condition proliferated slower and the culturedeteriorated due to the proliferation of differentiated cells. Recentstudies demonstrated that PD0325901 combined with CHIR99021 and FGFRinhibitor PD173074 can maintain mESC pluripotency in a LIF independentmanner (Ying et al., Cell 115, 281-292 (2003)). However, similar to thecombination of PD0325901and CHIR99021, including PD173074 (0.1 μM) inmedium didn't show further benefit. Because Activin A/Nodal signaling isimportant to maintain the undifferentiated state of hESCs and EpiSCs,but dispensable for mESC self-renewal, we then tested whether thecombination of PD0325901, CHIR99021 and the TGF-β inhibitor A-83-01 cansuppress the differentiation and promote self-renewal of riPSCs.Interestingly, with the combination of 0.5 μM PD0325901, 3 μM CHIR99021and 0.5 μM A-83-01, riPSCs grow as a more homogeneous population and thespontaneous differentiation was substantially inhibited (FIG. 1D). Undersuch condition, the clonal expansion efficiency was also significantlyincreased in comparison to the combination of PD0325901 and CHIR99021(FIG. 5). Furthermore, although LIF itself was not sufficient to sustainriPSC self-renewal, only very small ALP positive colonies were observedand could not be long-term maintained in the absence of LIF (FIG. 5).Moreover, the unique chemical inhibitor cocktail was required for longterm self-renewal of riPSCs. riPSCs have been cultured in the presenceof LIF, PD0325901, A-83-01, and CHIR99021 for more than 30 passageswithout obvious differentiation and decrease in proliferation, but theylose ESC morphology and differentiate within one passage after thechemical inhibitors are removed from the medium. Under this condition,riPSCs are similar to the conventional mESCs in forming typical domedcolonies in culture (FIG. 1E). Immunocytochemistry revealed that riPSCsexpress typical mESC markers, such as Oct4 (FIG. 1F), Sox2 (FIG. 1G),SSEA-1 (FIG. 1H, Green), Nanog (FIG. 1H Red), but are negative to thehESC markers, such as SSEA3, SSEA4 and TRA-1-81. RT-PCR analysis of fourclonal riPSC lines using rat gene primers confirmed the expression ofthe endogenous rat Oct4, Sox2, Nanog, Klf4, Rex-1, TDGF2, FGF4 and Eras(FIG. 1I). By using the specific primers for transgenes, RT-PCR analysisrevealed that the transduced mouse Oct4, Sox2 and Klf4 genes werelargely silenced (FIG. 1I). Analysis of the methylation status of therat Oct4 promoter showed differential methylation between riPSCs andWB-F344 cells. riPSC clones exhibited an almost complete demethylationpattern and is distinct from that of the parental WB-F344 cells (FIG.1J). Notably, riPSCs share common molecular features with mESCs,especially express Rex-1 and ALP, markers of ESCs and early epiblaststhat are absent in post-implantation stage epiblasts and EpiSCs.

The riPSCs are pluripotent stem cells in vitro and in vivo. To examinethe developmental potential of riPSCs, in vitro differentiation assaywas performed. Immunostaining showed riPSCs could differentiate intoendoderm (Albumin and Pdx1) (FIG. 2A, 2B), neuroectoderm (βIII-tubulin,Tuj1) (FIG. 2C) and mesoderm (brachyury) (FIG. 2D) derivatives understandard embryoid body differentiation methods. Next, we examinedriPSC's in vivo developmental potential. After transplanted into theSevere Combined Immunodeficient (SCID) mice, riPSCs formed teratoma,which consisted of all three germ layers including neuroepithelium-likestructure (ectoderm), airway epithelium (endoderm), cartilage-likestructure (mesoderm) and smooth muscle (mesoderm) (FIG. 2E-2H). Mostremarkably, after injected into Brown-Norway rat (black fur) blastocysts(n=18), three rats were born and all exhibited extensive coat-colorchimerism (FIG. 2I). However, no germline transmission has been detectedyet. Taken together, the above results defined our riPSCs, distinct fromrat EpiSCs, as a pluripotent mESC-like rat stem cell line.

Example 2

This example demonstrates culture media for inducing and maintaininghuman pluripotent cells in a state analogous to mouse embryonic stemcells.

In contrast to mESCs, bFGF and TGFβ/Activin/Nodal signaling areessential for maintaining self-renewal of the conventional hESCs andEpiSCs. Inhibition of either TGFβ/Activin/Nodal or FGFR signals causesdramatic and rapid differentiation of hESCs and EpiSCs under theconventional hESC culture conditions. Recently, human inducedpluripotent stem cells (hiPSCs) have been generated from fibroblasts byexpression of either Oct4, Sox2, c-Myc, and Klf4 or Oct4, Sox2, Nanog,and Lin28 under the bFGF culture condition (Dimos et al., Science 321,1218-1221 (2008); Lowry et al., Proc Natl Acad Sci USA 105, 2883-2888(2008); Nakagawa et al., Nat Biotechnol 26, 101-106 (2008); Takahashi etal., Cell 131, 861-872 (2007); Yu et al., Science 318, 1917-1920(2007)). Such hiPSCs closely resemble the conventional hESCs and EpiSCsin the signaling requirements for self-renewal and cell morphology.Based on the rat studies, we explored whether a mESC-like pluripotencystate for human pluripotent stem cells could be captured and maintained.Remarkably, we found hiPSCs could be effectively generated from IMR90human fibroblasts by viral expression of Oct4, Sox2, Nanog, and Lin28 inthe mESC medium containing hLIF with a similar timing and efficiency(FIG. 3A, 15-20 iPS cell colonies from 1×10⁵ transduced cells onaverage) as that under the conventional condition (Yu et al., Science318, 1917-1920 (2007)), and subsequently selected and expanded byaddition of the chemical cocktail of ALK5, GSK3 and MEK inhibitors. SuchhiPSCs long-term and homogenously self-renew (>20 passages) under hLIFand the chemical cocktail of PD0325901, A-83-01 and CHIR99021 (FIG. 3B).In contrast to the conventional hESCs, these hiPSCs formed ALP-positivedomed colonies similar to mESCs (FIG. 3C), were resistant to MEKinhibitor and ALK5 inhibitor, and whereas the conventional hESC line H1differentiated rapidly under the same conditions. These hiPSCshomogenously express typical pluripotency markers, such as Oct4, Sox2,Nanog, TRA-1-81, SSEA3 and SSEA-4 (FIG. 3D-3I). RT-PCR analysis of fourclonal hiPSC lines under such condition confirmed the expression of theendogenous human Oct4, Sox2, Nanog, Rex-1, TDGF2 and FGF4 (FIG. 3J). Byusing the specific primers for transgenes, RT-PCR analysis revealed thatthe transduced Oct4, Sox2 and Nanog genes were largely silenced (FIG.3J). Moreover, these hiPSCs showed similar DNA methylation patterns onOct4 promoter as H1 human ESCs and are distinct from the parental IMR90fibroblast (FIG. 3K). Similar to riPSCs, the inhibitor cocktail wasrequired for maintaining the domed colony morphology and long term invitro self-renewal of hiPSCs. Removing the inhibitor cocktail caused thecells to lose colony morphology within one passage. Removing hLIF fromthe medium did not show immediate/dramatic effects on the cells.However, hLIF seems to be useful for the long-term culture of hiPSCs.When cultured in the medium containing the chemical inhibitors butwithout hLIF, hiPSC culture deteriorates gradually and could not behomogenously passaged beyond 10 passages. Nevertheless, the exactsignaling mechanism in self-renewal of such novel hiPSCs remains to bedetermined.

Importantly, immunocytochemistry confirmed that such novel hiPSCs coulddifferentiate into endoderm (Albumin) (FIG. 4A), neuroectoderm(βIII-tubulin, Tuj1) (FIG. 4B) and mesoderm (brachyury) (FIG. 4C)derivatives in vitro. Furthermore, after transplanted into the SCIDmice, such hiPSCs formed teratoma, which consisted of all three germlayers including neuroepithelium-like structure (ectoderm) (FIG. 4D),epithelial tube structure (endoderm) (FIG. 4D), and cartilage-likestructure (mesoderm) (FIG. 4E). Taken together, the above resultssuggested that a mESC-like human pluripotent stem cell can be capturedand long-term maintained.

Discussion

Although embryonic stem cells have been established from mice since 1981(Martin, G. R., Proc Natl Acad Sci USA 78, 7634-7638 (1981)), attemptsto derive their counterparts from other animals such as rat, have notcompletely succeeded (Demers et al., Cloning Stem Cells 9, 512-522(2007); Ruhnke et al., Stem Cells 21, 428-436 (2003), 2003; Schulze etal., Methods Mol Biol 329, 45-58 (2006); Ueda et al., “Establishment ofrat embryonic stem cells and making of chimera rats,” PLoS ONE 3, e2800(2008)). By combining the genetic reprogramming and chemical approach,we were able to generate novel mESC-like rat and human pluripotent stemcells that share key characteristics of the conventional mESCs in colonymorphology and culture requirements/signaling responses. Under theunique cocktail of small molecules, our stable riPSCs were capable ofextensively contributing to chimerism in vivo. Rats are more suited forphysiological and behavioral studies and are excellent model formultigenic human diseases. However, the utilization of this invaluablemodel was hindered due to the unavailability of rat stem cells that arepluripotent in vivo. Our establishment of rat pluripotent cells andstrategy of using the appropriate chemical cocktails will pave the wayto generate gene-targeted rats for biomedical researches. Our hiPSCsgrow more robustly and seem to represent a novel pluripotent state thatis similar to the conventional mESCs and different from the conventionalhESCs.

Taken together, such findings underscore the unique advantage of thechemical approach, and pinpoint the importance of inhibiting TGF-βpathway for maintaining the mESC-like pluripotent state of rat and humancells. Our studies collectively provide a framework to generatepluripotent stem cells by reprogramming or derive early ICM-stage ESCsfrom rats or other species, whose embryonic stem cells are still notavailable.

Experimental Procedures

Cell culture and viral transduction: Diploid rat WB-F344 cells (Grishamet al., Proc Soc Exp Biol Med 204, 270-279 (1993)), a kind gift fromProf. William B. Coleman at University of North Carolina, at passage 7were transduced by pMXs-based retroviruses for mouse Oct4, Klf4 and Sox2(Addgene) as described (Takahashi, K., and Yamanaka, S., Cell 126,663-676 (2006)). 24 hours later, 1×10⁵ transduced WB-F344 cells wereseeded on the X-ray inactivated CF1 MEFs in 100 mm dish and incubatedwith mESC growth medium: Knockout™ DMEM, 20% Knockout serum replacement,1% Glutamax, 1% Non-essential amino acids, 1% penicillin/streptomycin,0.1 mM β-mercaptoethanol and 10³ U/ml mLIF (Millipore). After 10 days,the riPSC colonies were picked up for expansion on MEF feeder cells inmESC growth medium, and treated with MEK inhibitor PD0325901 (Stemgent,0.5 μM), ALK5 inhibitor A-83-01 (Tocris Bioscience, 0.5 μM), and GSK3βinhibitor CHIR99021 (Stemgent, 3 μM).

Human fibroblasts IMR90 (ATCC No. CCL-186) were cultured and transducedby pSin-EF2-Puro-based lentiviruses for human Oct4, Sox2, Nanog andLin28 (Addgene) as described (Yu et al., Science 318, 1917-1920 (2007)).24 hours later, 1×10⁵ transduced IMR-90 cells were seeded on the X-rayinactivated CF1 MEF feeder cells in a 100 mm dish. 24 hours later, themedia was changed to mESC medium supplemented with 10³ U/ml hLIF(Millipore). After three weeks, the hiPSC colonies were observed andpicked up at the fourth week post-infection for expansion on feedercells in the same medium containing MEK inhibitor PD0325901 (0.5 μM),ALK5 inhibitor A-83-01 (0.25 μM), and GSK3β inhibitor CHIR99021 (3 μM).

Blastocyst Injection: The blastocysts were recovered from the uterus ofBrown-Norway (BN) females at 4.5 days post coitum. The blastocysts wereplaced in a drop of HEPES under mineral oil. 8-15 riPSCs were injectedinto the blastocyst cavity using a microinjection pipette. Afterinjection, blastocysts were transferred to pseudo-pregnant recipientfemales. All animal procedures were in accordance with the guidelines ofthe National Institute of Health.

Supplemental Data

The Supplemental data includes the supplemental experimental procedures,one table and one figure.

Supplemental Experimental Procedures:

Differentiation of iPSCs in vitro: The in vitro differentiation ofriPSCs or hiPSCs was carried out by the standard embryoid bodydifferentiation methods. The riPSCs or hiPSCs were dissociated by 0.05%Trypsin-EDTA and cultured in ultra-low attachment 100-mm dish in DMEMmedium supplemented with 10% FBS to form embryoid body (EBs). The mediumwas changed every another day. One week later, the EBs were harvestedand transferred into Matrigel-coated 6-well plate in DMEM medium with10% FBS. Three to seven day later, the cells were fixed forimmunocytochemistry analysis. All cell culture products were fromInvitrogen/Gibco BRL except where mentioned.

Cytochemistry and immunofluorescence assay: Alkaline Phosphatasestaining was performed according to the manufacturer's protocol usingthe Alkaline Phosphatase Detection Kit (Millipore). Forimmunofluorescence assay, cells were fixed in 4% paraformaldehyde for 10minutes and washed three times with PBS containing 0.1% Triton X-100(Sigma-Aldrich). The fixed cells were then incubated in blocking buffer,0.1% Triton X-100 and 10% normal donkey serum (Jackson ImmunoResearchLaboratories Inc) in PBS (Invitrogen/Gibco BRL), for 30 min at roomtemperature (RT). The cells were then incubated with primary antibodyovernight at 4° C. in blocking buffer. The day after, cells were washedwith PBS and incubated with secondary antibody in PBS containing 0.1%Triton X-100 for one hour at RT. Mouse anti-Oct4 antibody (1:250) (SantaCruz Biotechnology), rabbit anti-Sox2 antibody (1:2000) (Chemicon),mouse anti-SSEA1 antibody (1:250) (Santa Cruz Biotechnology), rabbitanti-Nanog antibody (1:500) (Abcam), rat anti-SSEA3 antibody (1:1000)(Chemicon), mouse anti-SSEA4 antibody (1:1000) (Chemicon), mouseanti-TRA-1-81 antibody (1:1000) (Chemicon), rabbit anti-Pdx1 (1:1500), agift from Dr. C. Wright (Vanderbilt University, TN), mouse anti-(Tuj1)antibody (1:1000) (Covance Research Products), rabbit anti-albuminantibody (1:1000) (DAKO) were used as primary antibodies. Secondaryantibodies were Alexa Fluor 486/555 donkey anti-mouse, anti-rat,anti-goat or anti-rabbit IgG (1:500) (Invitrogen). Nuclei werevisualized by DAPI (Sigma-Aldrich) staining. Images were captured usinga Nikon Eclipse TE2000-U microscope.

RT-PCR analysis: RNA was extracted from riPSCs and hiPSCs using theRNeasy Plus Mini Kit in combination with QIAshredder (Qiagene). Reversetranscription was performed with 1 μg RNA using iScript™cDNA SynthesisKit (BioRad). Amplification of specific genes was done using primersshowed in Table 1. The PCR conditions were 95° C. for 5 minutes, 94° C.for 30 seconds, annealing temperature for 30 seconds, and 72° C. for 30seconds, 25˜35 cycles, and then 72° C. for 10 minutes. For Oct4 promotermethylation study using bisulfite-sequencing, DNAs from WB-F344 cells,riPSCs, IMR90, and hiPSCs were isolated using the Non Organic DNAIsolation Kit (Millipore). The DNAs were then treated with the EZ DNAMethylation-Gold Kit (Zymo Research Corp., Orange, Calif.). The treatedDNAs were then used as templates to amplify sequences of interest.Primers used for Oct4 promoter fragment amplification were showed inTable 1. The resulting fragments were cloned using the TOPO TA CloningKit for sequencing (Invitrogen) and sequenced.

Teratoma Formation: The serially passaged riPSCs or hiPSCs wereharvested by using 0.05% Trypsin-EDTA. Three to five million cells wereinjected under the kidney capsule of SCID mice (n=3). After 4-5 weeks,all mice developed teratomas, which were removed and then histologicallyanalyzed.

TABLE 1 List of primers information for PCR Forward primer Reverse primer  Size Genes (SEQ ID NO:) (SEQ ID NO:) (bp)For RT-RCR (rat cells) Rat Oct4 TACTGCCCGCCCCAGCG GCTGCTTGGCAATGCTAGT449 (2) (3) Rat Sox2 AAGGCCGTGCACGCCGA ACCACACCATGAAGGCATTC 285 CGA (4)AT (5) Rat Nanog TAGCCCTGATTCTTCTA TTTGCTGCAACGGCACATAA 617 GCA (6) (7)Rat Rex-1 AAATCATACGAGGCA TGAGTTCGCTCCAACAGTCT 350 AGGC (8) (9) Rat Klf4CAGACCTGGAAAGTGGT ACCTGTGTTGCCCGCAGCC 283 GG (10) (11) Rat TDGF2AACACCAACAATATTTT TCATTTCTAGGAAAAGGCAG 511 ATGTGGCC (12) ATGC (13)Rat FGF-4 TGTGGTGAGCATCTTCG CCTTCTTGGTCCGCCCGTTC 198 GAGTGG (14)TTA (15) Rat Eras GCTGCCCCTCAGCCGAC CACTGCCTTGTACTCCGGTA 210TGCTACT (16) GCTG (17) Transgenic GGGGTGGACCATCCTCT CCTCCGCAGAACTCGTAT271 Oct4 A (18) (19) Transgenic CCCACCGCCCTCAAAGT GGACCATACCATGAAGGCG278 Sox2 A (20) TT (21) Transgenic CCCACCGCCCTCAAAGT GCTGGACGCAGTGTCTTCT190 K1f4 A (22) (23) GADPH CCTTCATTGACCTCAAC GGAAGGCCATGCCAGTGAG 594TAC (24) C (25) For RT-RCR (human cells) Endogenous AGTTTGTGCCAGGGTTTACTTCACCTTCCCTCCAACC 113 Oct4 TTG (26) (27) Endogenous TTTGGAAGCTGCTGGGGGATGGGAGGAGGGGAGAGG 194 Nanog AAG (28) A (29) EndogenousCAAAAATGGCCATGCAG AGTTGGGATCGAACAAAAG 162 Sox2 GTT (30) CTATT (31) Rex-1CAGATCCTAAACAGCTC GCGTACGCAAATTAAAGTCC 307 GCAGAAT (32) AGA (33) FGF-4CTACAACGCCTACGAGT GTTGCACCAGAAAAGTCAG 369 CCTACA (34) AGTTG (35) TDGF2CTGCTGCCTGAATGGGG GCCACGAGGTGCTCATCCAT 242 GAACCTGC (36) CACAAGG (37)Transgenic CAGTGCCCGAAACCCAC AGAGGAACTGCTTCCTTCAC 656 Oct4 AC (38)GACA (39) Transgenic TACCTCTTCCTCCCACTC AGAGGAACTGCTTCCTTCAC 467 Sox2CA (40) GACA (41) Transgenic CAGAAGGCCTCAGCACC AGAGGAACTGCTTCCTTCAC 732Nanog TAC (42) GACA (43) GADPH GTGGACCTGACCTGCCG GGAGGAGTGGGTGTCGCTG 152TCT (44) T (45) For bi sulfite-sequencing Rat Oct4 ATGGGATTTTGGAGGATCTCAAACCCAAATACCCCTA 206 TTTTAG (46) CTT (47) Human GGATGTTATTAAGATGACCTAAACTCCCCTTCAAAAT 406 Oct4 AGATAGTTGG (48) CTATT (49)

Example 3 Derivation of Rat Embryonic Stem Cells

To derive rat ES cells from blastocyst, zona pellucida-removed ratblastocysts (E4.5) are seeded on x-ray inactivated CF1 feeder cells withthe Knock-out DMEM medium supplemented with 20% Knock-out serumreplacement (KSR), 1% non-essential amino acid, 1000 U/ml mouse LIF, 1%Glutmax, 3 μM CHIR99021, 0.5 μM PD0325901, 0.25 μM A-83-01 (or 2 μMSB431542). After 3-5 days, the ICM derived cell clumps were dissociatedby Accutase and transferred onto the new feeders. Colonies with typicalES cell morphology were picked up, dissociated with Accutase and thenseeded on new feeders. The established rat ES cells were cultured withabove medium and passaged about every 3 days (1:6). Both rat ES cellsand riPSCs require the presence of LIF and inhibitor cocktail(CHIR99021, PD0325901, A-83-01) for long-term self-renewal. Rat ES cellsand riPSCs express the pluripotent markers of mouse ES cells, such asOct4, Sox2, Nanog and SSEA-1, etc., but not the markers, such as SSEA-3,SSEA-4, TRA-1-61 and TRA-1-81, that were expressed byconventionally-cultured human ES cells. Both rat ES cells and riPSCsexpress the ICM markers Rex-1 and ALP, which are not expressed in mouseEpiSCs.

Example 4 Derivation of Human and Monkey Embryonic Stem Cells

To convert human and monkey ES cells to mouse ES cell-like (early ICM)pluripotent state, human ES cells (Hues9) and Monkey ES cells (R366.4)were cultured with the DMEM/F-12 medium supplemented with 20% Knock-outserum replacement (KSR), 1% non-essential amino acid, 1% Glutmax, 10ng/ml bFGF. When the cells get to 50% confluence, the media wereswitched to Advance DMEM/F-12, 1×N2, 1×B27, 1% Glutmax, 50 μg/ml BSAmedium supplemented with 2 μM Lysine-Specific Demethylase 1 inhibitor(Parnate). After three days, the cells were cultured with the samemedium containing 10 μg/ml human LiF, 3 μM CHIR99021, 0.5 μM PD0325901,and 2 μM SB431542, but without Parnate. Despite the extensivedifferentiation, the compact mouse ES cell-like colonies were visualizedabout one week treatment. The converted human/monkey ES cells werecultured with above medium and passaged about every 4-5 days (1:6). Thecells express the pluripotent markers, such as Oct4, Sox2, Nanog,SSEA-3, SSEA-4, TRA-1-61, TRA-1-81 and also the ICM markers Rex-1 andALP.

Example 5 Conversion of Epiblast Stem Cells to Embryonic Stem Cells bySmall Molecules

Conventional murine embryonic stem cells (ESCs) are derived from andrepresent pluripotent cells of the inner cell mass (ICM) ofpre-implantation blastocysts. They can self-renew indefinitely and havethe ability to give rise to all cell types in vitro, and mostimportantly contribute to an entire animal in vivo, including germline,when placed back into blastocysts. More recently, a different type ofpluripotent cells was derived from post-implantation stage epiblasts,termed epiblast stem cells (EpiSCs) (Brons et al., Nature 448, 191-195,2007; Tesar et al., Nature 448, 196-199, 2007). While EpiSCs canlong-term self-renew and appear to be pluripotent in vitro as well as invivo in teratoma assays, in contrast to mESCs, they are incapable ofincorporating into ICM and contributing to chimerism, confirming thatEpiSCs are from and represent an advanced/later developmental stage ofpluripotency than ICM-derived ESCs and suggesting they could not be“reprogrammed” back into ICM-stage pluripotent cells even under the invivo environment. Conventional human ESCs, although derived usingblastocysts, seem to correspond very closely to the EpiSCs with respectto many characteristics, including some gene expression, colonymorphology (i.e. flat colony) and the signaling responses inself-renewal and differentiation. EpiSCs/hESCs are also functionally andmechanistically distinct from mESCs (which have more compact and domedcolony morphology) in many other ways. For example, while mESCsself-renew under leukemia inhibitory factor (LIF) and bone morphogenicprotein (BMP) (Ying et al., Cell 115, 281-292, 2003), or underinhibition of MEK and/or FGFR (Ying et al., Nature 453, 519-523, 2008),EpiSCs/hESCs appear dependent on MAPK, FGF, and TGFβ/Activin/Nodalpathway activity for self-renewal, and differentiate rapidly whentreated with MEK, FGFR and/or ALK4/5/7 inhibitors (Brons et al., Nature448, 191-195, 2007; Li et al., Differentiation 75, 299-307, 2007;Peerani et al., EMBO J 26, 4744-4755, 2007; Tesar et al., Nature 448,196-199, 2007). In addition, in response to BMP treatment under defineddifferentiation conditions, mESCs differentiate toward mesoderm lineageswhile EpiSCs/hESCs generate trophoblasts or primitive endoderm cells(Brons et al., Nature 448, 191-195, 2007; D'Amour et al., Nat Biotechnol23, 1534-1541, 2005; Xu et al., Nat Biotechnol 20, 1261-1264, 2002).These observations strongly support the notion that EpiSCs and hESCs areintrinsically similar, and raise an attractive hypothesis: as mESCs andEpiSCs/hESCs represent two distinct pluripotency states: the mESC-likestate representing the ICM of pre-implantation blastocyst and theEpiSC-like state representing the post-implantation epiblasts, whetherthe epiblast state (including conventional hESCs) can be converted backto the ICM state. Because of the distinct difference in their ability tocontribute to chimerism from mESCs or mEpiSCs (which would offer adefinitive confirmation of functional conversion of EpiSCs to mESCs),the murine system represents an ideal platform to study such anintriguing process, and provides a basis for generating perhaps a newtype of ICM/mESC-like human pluripotent cells from conventional hESCs.

EpiSCs express master pluripotency genes, including Oct4, Sox2 andNanog. Overexpression of Oct4, Sox2 and Klf4 has been shown to inducereprogramming of murine somatic cells to become germline-competentpluripotent cells (Nakagawa et al., Nat Biotechnol 26, 101-106, 2008).In addition, it has been shown that germline stem cells, which expressfewer pluripotency genes (e.g. lack of Nanog expression), can convert tomESC-like cells in culture (Chambers et al., Nature 450, 1230-1234,2007; Kanatsu-Shinohara et al., Cell 119, 1001-1012, 2004). Furthermore,recently a non-pluripotent cell type (called FAB-SC) was derived fromblastocytes, and was shown to generate pluripotent mESC-like cellssimply under LIF and BMP condition (Chou et al., Cell 135, 449-461,2008). Moreover, recent studies suggested sub-populations of cellswithin mESC colonies exhibited dynamic expression of several keytranscription factors (e.g. Nanog, Rex1, and Stella) that makes themfluctuate between different states continuously (e.g. between an ESC-and epiblast-like phenotypes) (Chambers et al., Nature 450, 1230-1234,2007; Hayashi et al., Cell Stem Cell 3, 391-401, 2008; Singh et al.,Stem Cells 25, 2534-2542, 2007; Toyooka et al., Development 135,909-918, 2008). These studies raise the possibility that EpiSCs existingin a less “stable” pluripotency state than ICM-mESCs may have theability to transition back to a mESC state “spontaneously” under culturefluctuation in vitro. To test this hypothesis, EpiSCs were trypsinizedto single cells and plated under mESC self-renewal conditions, based onthe notion that “converted” mESC-like cells within EpiSC colonies wouldbe captured/selected and expanded under the conditions that promoteself-renewal of mESCs but induce differentiation of EpiSCs. We foundEpiSCs differentiated (e.g. cells spread/migrated out of colonies) inthe first passage and no colony could be identified over severalpassages when they were cultured under the conventional mESC growthcondition with feeder cells and supplemented with LIF (FIG. 6A). Giventhat the “spontaneous” conversion from EpiSCs to mESCs might be veryinefficient, a stronger and more stringent differential self-renewalpromoting and differentiation inducing condition might be required toselect/capture and expand those “rare” converted mESC-like cells fromEpiSCs (e.g. achieving cleaner phenotypic distinction and minimizing theovergrowth of differentiated EpiSCs). Based on the differentialsignaling responses (self-renewal vs. differentiation) between mESCs andEpiSCs in the context of FGF and MAPK signaling pathways, as well as theobservation that inhibition of MEK-ERK signaling promotes reprogrammingof cells towards more primitive state (Chen et al., Proc Natl Acad SciUSA 104, 10482-10487, 2007; Shi et al., Cell Stem Cell 2, 525-528, 2008;Silva et al., PLoS Biol 6, e253, 2008), we next treated EpiSCs with acombination of selective FGFR inhibitor PD173074 (0.1 μM) and MEKinhibitor PD0325901 (0.5 μM) under the regular mESC self-renewalcondition. Under this 2PD/LIF condition that promotes robust clonalgrowth of mESCs and inhibits growth of differentiated cells, we observedaccelerated differentiation of EpiSCs and decreased growth of overallcell culture. Most of cells died when kept culturing in the 2PD/LIFmedium and no mESC-like colony was identified over serial passages.Similarly, adding CHIR99021 (3 μM) to the 2PD/LIF condition for improvedmESC growth/survival did not promote or capture the conversion of EpiSCsto mESC-like state (FIG. 6A). These results suggested that the EpiSCsrepresent a “stable” pluripotency state that does not readily convert toan ESC-like state spontaneously under conditions promoting mESCself-renewal. This is also consistent with a more recent study where itwas shown that conversion of EpiSCs to mESC-like state could only beachieved by overexpression of Klf4 in conjunction with using chemicalinhibitors of MEK and GSK3 (Guo et al., Development 136, 1063-1069,2009).

TGFβ/Activin/Nodal activity is dynamically regulated temporally andspatially during mouse embryogenesis and is required during implantationto control fate of early progenitor cells in the epiblasts (Mesnard etal., Development 133, 2497-2505, 2006). The derivation of EpiSCsrequiring FGF and TGFβ/Activin/Nodal pathway activities suggests thatTGFβ/Activin/Nodal provides an anti-differentiation signal for EpiSCs(Brons et al., Nature 448, 191-195, 2007; Tesar et al., Nature 448,196-199, 2007). In addition, it was reported that E-cadherin isexpressed in embryos from the one-cell-stage, and down-regulation ofE-cadherin by signaling facilitates the implantation of blastocyst (Liet al., J Biol Chem 277, 46447-46455, 2002). Moreover,TGFβ/Activin/Nodal activities also promote epithelial-mesenchymaltransition (EMT) by down-regulating E-cadherin during gastrulation(Derynck and Akhurst, Nat Cell Biol 9, 1000-1004, 2007; Gadue et al.,Proc Natl Acad Sci USA 103, 16806-16811, 2006; Sirard et al., Genes Dev12, 107-119, 1998). Based on these studies, we hypothesized thatinhibition of TGFβ/Activin/Nodal signaling might promote the process ofmesenchymal-epithelial transition (MET) and consequently the conversionof EpiSC to the mESC-like state. A-83-01 is a selective ALK4/5/7inhibitor, which has no cross inhibitory effect on the BMP receptors(Tojo et al., Cancer Sci 96, 791-800, 2005). Consistent with theprevious reports, blocking TGFβ/Activin/Nodal signaling by 0.5 μMA-83-01 induced rapid differentiation of EpiSCs under EpiSC/hESC culturecondition that is supplemented with bFGF. In dramatic contrast, undermESC culture condition that is supplemented with LIF, A-83-01 inducedoverall population of EpiSCs to form more compact and domed coloniesthat resemble mESC colony morphology and express ALP (a pluripotencymarker highly expressed in mESCs, but not in EpiSCs) (FIG. 6C). Anotherwidely used specific ALK4/5/7 inhibitor SB431542 has a similar effect onEpiSCs. When the A-83-01 treated colonies were exposed to 2PD/LIFcondition for selection, more than 50% of colonies could self-renew andmaintain ALP activity, suggesting the cells acquired some mESCproperties. Those domed ALP positive colonies were further maintainedand expanded in mESC growth media supplemented with inhibitors of ALK5,MEK, FGFR and GSK3 (named mAMFGi condition). These cells can long termself-renew under the mAMFGi condition, have an indistinguishable mESCcolony morphology (FIG. 6D), and express pluripotency markers such asOct4, Nanog, SSEA-1, as well as regain the ICM marker Rex-1. However,when these cells were labeled with a constitutively active GFP bylentiviruses, and aggregated with morulas, we did not obtain chimericanimals after the resulted embryos were transplanted into mice (FIG.7A). These results indicated that inhibition of TGFβ signaling inconjunction with inhibition of MEK, FGFR and GSK3 has strongreprogramming activity and can promote partial conversion of EpiSCs to amESC-like state.

Histone modifications, such as acetylation and methylation, have beenestablished to play important roles in gene regulation. It has beenindicated that Stella is an important gene in mESC's competence togermline, and is transcriptionally silent in EpiSCs and epiblast-likecells within mESCs. Moreover, histone modification regulates the Stellaexpression in mESCs (Hayashi et al., Cell Stem Cell 3, 391-401, 2008;Tesar et al., Nature 448, 196-199, 2007). We hypothesized that aderepression of the silenced gene loci responsible for true in vivopluripotency may promote EpiSCs to overcome the epigeneticrestriction/threshold toward mESC-like state. Consequently, we chose thesmall molecule parnate, which has been shown to increase global H3K4methylation by inhibiting the histone demethylase LSD1 that specificallydemethylates mono- and di-methylated histone H3K4 (Lee et al., Chem Biol13, 563-567, 2006). Remarkably, after four days of 2 μM parnatetreatment, up to 70-80% of the EpiSCs formed small and compact coloniesin the mESC growth condition. When the parnate-treated cells were thenselected with 2PD/LIF, roughly 20% of cells survived the selection asdomed and ALP positive colonies. Those colonies were further expandedwith inhibitors of MEK, FGFR and GSK3 (named mMFGi condition) or withthe mAMFGi condition. Both conditions resulted in a stable cell culture(>80 passages over 8 months), that is morphologically indistinguishablefrom mESCs (FIG. 6E, F). We next examined GFP-labeled parnate/mMFGicells and parnate/mAMFGi cells in vivo by morula aggregation andtransplantation of resulted embryos. Remarkably, we obtained 7 (out of 9born pups) adult chimeras from parnate/mAMFGi cells as determined bycoat color and PCR genotyping for the presence of GFP integration inmultiple adult tissues (FIG. 7A, B, C). Consistently, widespread GFPpositive cells were observed in multiple tissues (i.e. three germlayers, including gonad) of E13.5 embryos from transplantation of theparnate/mAMFGi cell-aggregated morulas (FIG. 7A, D, 10A). To examinegermline contribution from parnate/mAMFGi cells, the GFP/SSEA-1 doublepositive cells from the gonad were isolated by FACS and confirmed toexpress germ line markers Blimp1 and Stella by real-time PCR (FIG. 7E).These data suggest that parnate/mAMFGi cells converted from EpiSCsregain true in vivo pluripotency. In contrast, GFP-positive cells wereonly found in the yolk sac of E13.5 embryos recovered fromtransplantation of parnate/mMFGi cell-aggregated morulas (FIG. 7A).

The parnate/mAMFGi cells were therefore further characterized.Immunocytochemistry confirmed homogeneous expression ofpluripotency-associated markers in long-term expanded parnate/mAMFGicells, including Oct4, Nanog, SSEA1, and STELLA (FIG. 8A, 11A, 12). Inaddition, semi-quantitative RT-PCR analysis demonstrated restoration ofgene expression of specific ICM and germline-competence markers (thatare expressed in mESCs, but absent in EpiSCs) in parnate/mAMFGi cells,including Rex1, Pecam1, Stella, Stra8, Dax1, Fbxo15, Esrrb, and Fgf4(FIG. 8B). In contrast, transcripts of genes associated with theepiblast and early germ layers such as Fgf5 and Brachyury (T) weredecreased or undetectable in parnate/mAMFGi cells (FIG. 8B, 9C).Furthermore, transcriptome analysis by microarray demonstrated that theconverted parnate/mAMFGi cells are much more similar to mESCs (Pearsoncorrelation value: 0.87), while the original EpiSCs are more distantfrom mESCs (Pearson correlation value: 0.74) (FIG. 8C), consistent withprevious reports. To further analyze specific epigenetic changesassociated with the conversion, we examined the promoter DNA methylationof Stella and Fgf4, whose expressions are closely associated with ICMproperties (Hayashi et al., Cell Stem Cell 3, 391-401, 2008; Imamura etal., BMC Dev Biol 6, 34, 2006), using bisulphite genomic sequencing. Itrevealed that the promoter regions of Stella and Fgf4 were largelyunmethylated in parnate/mAMFGi cells and mESCs, but were hypermethylatedin EpiSCs (FIG. 8D). To further examine the epigenetic state of Stella,which is restricted to the mESC-like state, we performed a ChIP-QPCRanalysis of its promoter region in EpiSCs, converted parnate/mAMFGicells, and mESCs. We found that the H3K4 and H3K27 methylation patternof Stella in parnate/mAMFGi cells is similar to that observed in mESCs,but is distinct from that in EpiSCs, confirming that the epigeneticstatus of Stella in the converted parnate/mAMFGi cells was switched tothe mESC-like status (FIG. 8E).

Parnate/mAMFGi cells were also examined for their in vitro functionalproperties. They were found to have similar growth rate as mESCs (FIG.9A). When Parnate/mAMFGi cells were differentiated through embryoidbodies in suspension, they were able to effectively generate cellderivatives in the three primary germ layers as shown byimmunocytochemistry, including characteristic neuronal cells(βIII-tubulin and MAP2ab positive), beating cardiomyocytes (cardiactroponin and MHC positive), and endoderm cells (Sox17 or Albuminpositive) (FIG. 9B. Because mESCs and EpiSCs/hESCs have differentresponses to signaling inputs (e.g. growth factors) in self-renewal anddifferentiation, conditions that were developed and work effectively formESC differentiation may often be inefficient in inducing correspondingdifferentiation of EpiSCs/hESCs. One of the advantages for convertingEpiSC/hESC to a mESC-like state is that differentiation conditions maybe more readily translated from mESC work to EpiSC/hESC work.Differential response to BMP4 treatment represents a functional assay todistinguish between mESCs and EpiSCs. Consistent with the previousstudies (Beddington and Robertson, Development 105, 733-737, 1989; Czyzand Wobus, Differentiation 68, 167-174, 2001; Qi et al., Proc Natl AcadSci USA 101, 6027-6032, 2004; Winnier et al., Genes Dev 9, 2105-2116,1995), we found that parnate/mAMFGi cells were induced to express themesoderm specific marker gene Brachyury (T) when treated with BMP4 asmESCs, but under the same condition couldn't give rise to trophectoderm(no induction of trophoblast marker Cdx2) or primitive endoderm cells(Gata6) as EpiSCs, suggesting a similar in vitro differentiationpotential/response of parnate/mAMFGi cells to mESCs (FIG. 9C). Tofurther demonstrate this, we attempted and compared EpiSCs, convertedparnate/mAMFGi cells, and mESCs in a monolayer chemically defineddirected step-wise cardiac differentiation process. In this multi-stepprocess, where BMP activity plays an essential role in the early stepsof mesoderm differentiation, we found that parnate/mAMFGi cellsdifferentiated into beating cardiomyocytes as efficiently as mESCs, butdifferentiation of EpiSCs under the same condition hardly produced cellsthat expressed appropriate cardiac markers or have characteristicbeating phenotype (FIG. 9D), confirming again that parnate/mAMFGi cellsare functionally similar to mESCs. Moreover, a single cell survivalassay also demonstrated that parnate/mAMFGi cells clonally expand asOct4-positive colonies as efficiently as mESCs in feeder-free andN2/B27-chemically defined conditions, while EpiSCs survive poorly fromsingle cells under the same condition (FIG. 11B). These data furtherdemonstrated that EpiSCs could be functionally converted to themESC-like state by pharmacological manipulation that targets epigeneticmodifications and differential signaling pathways required by mESCs orEpiSCs.

Concurrent with our studies, EpiSC cells have recently been reported toconvert to a mESC-like state by overexpression of reprogramming genes(i.e. Klf4) in conjunction with chemical compounds (Guo et al.,Development 136, 1063-1069, 2009). In this study, we devised achemically defined treatment to convert stable EpiSCs to a mESC-like,developmentally earlier pluripotency state without any geneticmanipulation. Despite studies providing evidence that epiblast-likecells exist and transition back and forth within colony of conventionalmESCs (Hayashi et al., Cell Stem Cell 3, 391-401, 2008); mESCs andEpiSCs share substantial set of pluripotency transcriptional factors,including Oct4, Sox2 and Nanog; and mESCs are more stable in culture, inthe present study we found that EpiSCs differentiated rapidly under theconventional mESC culture conditions and no “spontaneously” convertedmESC could be readily identified and isolated over serial passages atthe population or clonal level. Remarkably, we found that blockage ofthe TGFβ pathway or inhibition of the H3K4 demethylase LSD1 with smallmolecule inhibitors induced dramatic morphological changes of EpiSCstowards mESC-like phenotypes with activation of some ICM-specific geneexpression. However, full conversion of EpiSCs to a mESC-like state withcompetence to chimeric contribution can only be readily generated with acombination of inhibitors of LSD1, ALK5, MEK, FGFR, and GSK3. Theseobservations underscore a powerful and direct induction of reprogrammingfrom the developmentally later-stage EpiSCs to the ICM-stage mESCs by asynergy of signaling and direct epigenetic modulations. It alsohighlights a significant role for TGFβ pathway inhibition in promotingreprogramming and sustaining true pluripotency, which further supportsour recent studies in generating chimerism-competent rat pluripotentcells (Li et al., Cell Stem Cell 4, 16-19, 2009). Collectively, ourstudies provide a proof-of-concept demonstration thatpluripotency-restricted EpiSCs can be readily converted to a mESC-likestate in the absence of any genetic manipulation by precisepharmacological control of signaling pathways that distinguish the twopluripotency states and an epigenetic target simultaneously, and offer aconvenient experimental system to further study the mechanism. Suchmethod and concept may also provide an avenue for generating a new typeof mESC-like human pluripotent cell.

Experimental Procedures

Cell culture: The murine EpiSC line was a gift from Dr. Paul Tesar (CaseWestern Reserve University). EpiSCs (line EpiSC-5, male) were maintainedon irradiated CF1 MEFs in human ESC medium supplemented with 10 ng/mlbFGF as described previously (Tesar et al., Nature 448, 196-199, 2007).EpiSCs were passaged every 3-4 days with 1 mg/ml collagenase type IV(Invitrogen). The R1 mESCs were cultured on irradiated CF1 MEFs withconventional mESC growth media, which consist of Knockout DMEM(Invitrogen) supplemented with 20% KSR (Invitrogen), 0.1 mM 2-ME(Sigma-Aldrich), 2 mM L-glutamine (Invitrogen), 0.1 mM NEAA(Invitrogen), and 10³ units/ml recombinant murine leukemia inhibitoryfactor (LIF) (ESGRO, Millipore). The mESCs and converted cells werepassaged every 3 days as a single cell suspension using 0.05%trypsin/EDTA and seeded at 1.0×10⁴ cells per cm² for routine culture.For feeder-free culture, cells are grown on gelatin-coated tissueculture dishes in chemically defined media, which consist of KnockoutDMEM supplemented with 1×N2 (Invitrogen), 1×B27 (Invitrogen), 0.1 mM2-ME, 2 mM L-glutamine, 0.1 mM NEAA, 50 μg/ml BSA fraction V (GIBCO),10³ units/ml LIF and 10 ng/ml BMP4 (R&D). For growth curve experiment,cells were cultured in the feeder-free condition in gelatin-coated12-well plates. Duplicate samples of cells were plated at a density of1×10⁵ cells per well. For each time point (24 hr apart), cells fromduplicate wells were trypsinized and counted using hemocytometer. Thosecounts were averaged, and plotted. ALK inhibitor A-83-01, SB431542, MEKinhibitor PD0325901, GSK3 inhibitor CHIR99021, and FGF receptorinhibitor PD173074 were purchased from Stemgent Inc. Parnate waspurchased from Sigma (P8511).

Semi-quantitative RT-PCR and real-time PCR: Total RNA were extracted byusing RNeasy plus mini kit (Qiagen), reverse transcribed with iScriptcDNA Synthesis Kit (Bio-Rad) using oligo dT primers according tomanufacturer instructions. PCR products were resolved on (1.5%) agarosegels and visualized by ethidium bromide staining. Images were takenusing Bio-Rad Gel document system. Diluted cDNA was used in each ofduplicate quantitative PCRs on a Bio-Rad real-time PCR detection systemwith IQ SYBR Green (Bio-Rad). Primers used are listed in SupplementalTable 2.

Bisulfite sequencing analysis: DNAs from R1 mESCs, EpiSCs, andParnate/mAMEGi cells were isolated using the Non Organic DNA IsolationKit (Millipore). The DNAs were then treated for bisulfate sequencingwith the EZ DNA Methylation-Gold Kit (Zymo Research Corp., Orange,Calif.). The treated DNAs were then used to amplify sequences ofinterest. Primers used for promoter fragment amplification were aspreviously published (Hayashi et al., Cell Stem Cell 3, 391-401, 2008;Imamura et al., BMC Dev Biol 6, 34, 2006) and listed in SupplementalTable 2. The resulting fragments were cloned using the TOPO TA CloningKit for sequencing (Invitrogen) and sequenced.

Flow cytometry and cell sorting: Adherent cells were washed twice in PBSand then incubated for 20 minutes at 37° C. in Cell Dissociation Buffer(Invitrogen). Cells were dissociated and re-suspended in PBS+3% normalgoat serum (blocking buffer). Cells were incubated for 40 minutes at 4°C. with antibody anti-SSEA1 (1:50, Santa Cruz) and then incubated withthe corresponding secondary antibody followed by washing steps. Cellswere analyzed and sorted using a FACSAria cell sorter and FACSDivasoftware (BD Biosciences). Using a 488-nm laser for excitation, GFPpositivity was determined according to fluorescence intensity in the GFPchannel. SSEA-1 positivity was determined according to fluorescence inthe red channel.

In vitro differentiation: Parnate/mAMFGi cells were trypsinized intosingle cells and cultured in suspension to form embryoid bodies/EBs inlow adhesion plates (Corning) in DMEM medium supplemented with 10% FBS.Media were refreshed every other day and EBs were allowed to grow for 6days in suspension. EBs were then replated onto 0.1% gelatin-coatedplates. Spontaneous differentiations were examined by immunostaining ofrepresentative lineage specific markers with indicated antibodies atvarious time points (3 up to 16 days). For directed cardiacdifferentiation, cells were plated on Matrigel coated plates at2×10⁴/cm². 24 hours after plating, cells are switched into chemicallydefined medium (CDM) [consisting of RPMI 1640, 0.5× N2, 1× B27 (withoutVitamin A), 0.5× Glutamax, 0.55 mM beta-mercaptoethanol, and 1×non-essential amino acids], and treated with 3 μM BIO (Calbiochem) and20 ng/ml BMP-4 (R&D) for five days. Then, the medium is changed to CDMcontaining 100 ng/ml Dkk-1 and cells are cultured for additional fivedays. At Day 11, cells are briefly trypsinized/detached (0.05% trypsin)and replated onto gelatin coated 6-well plates in CDM with no additionalgrowth factors, and cultured for additional 4-6 days when beatingphenotype appears in most of cardiac colonies.

Characterization assays: ALP staining was performed using the AlkalinePhosphatase Detection Kit (Chemicon) as instructed by the manufacturer.Immunocytochemistry was performed using standard protocol. Briefly,cells were fixed in 4% paraformaldehyde (Sigma-Aldrich), washed threetimes by PBS, and then incubated in PBS containing 0.3% TritonX-100(Sigma-Aldrich) and 5% normal donkey serum (Jackson Immuno Research) for1 hr at room temperature. The cells were then incubated with primaryantibody at 4° C. overnight: Albumin (Abcam, AB19188, 1:200); Brachyury(Santa Cruz, C-19, 1:200); Cardiac troponin t antibody (CT3)(Developmental Studies Hybridoma Bank, 1:700); MAP2ab (Abcam, ab5392,1:1000); MF20 (Developmental Studies Hybridoma Bank, 1:200); Nanog(Abcam, ab21603, 1:500); Oct4 (Santa Cruz, sc-5279, 1:100); Sox17 (R&Dsystems, AF1924, 1:300); SSEA1 (Santa Cruz, sc-21702, 1:100); Stella(Millipore, MAB4388, 1:200); Tuj-1 (Covance, MMS-435P, 1:1000). Afterwashing three times with PBS, cells were incubated with appropriateAlexa Fluor conjugated secondary antibodies (Invitrogen) for 2 hr at RT.Nuclei were detected by DAPI (Sigma) staining. Images were captured byZeiss HXP 120.

Chimera formation: The converted cells were stably marked by GFP usinglentiviruses. Cells were aggregated with 8-cell-stage mouse embryos, andwere then transplanted into the uteri of 2.5 dpc pseudo-pregnant CD1mice.

Microarray analysis: The Illumina Sentrix BeadChip Array MouseRef-8 v2(Illumina, Calif., USA) was used for microarray hybridizations toexamine the global gene expression of murine ES cells, EpiSCs cells andParnate/mAMFGi cells. Biotin-16-UTP-labeled cRNA was synthesized from500 ng total RNA with the Illumina TotalPrep RNA amplification kit(Ambion AMIL1791, Foster City, Calif., USA). The hybridization mixcontaining 750 ng of labeled amplified cRNA was prepared according tothe Illumina BeadStation 500× System Manual (Illumina, San Diego,Calif., USA) using the supplied reagents and GE HealthcareStreptavidin-Cy3 staining solution. Hybridization to the Illumina ArrayMouseRef-8 v2 was for 18 h at 55° C. on a BeadChip Hyb Wheel. The arraywas scanned using the Illumina BeadArray Reader. All samples wereprepared in two biological replicates. Processing and analysis of themicroarray data were performed with the Illumina BeadStudio software.The microarray data of R1-mESC cells was from our previous studies GEODataSet (GSM402334 and GSM402335). All raw data were subtracted forbackground and normalized together using the rank invariant option. Wehave deposited the microarray data of parnate/mAMFGi and EpiSCs to GEODataSets with the accession number (GSM402334 and GSM402335 for R1 mouseES cells; GSE17664 for parnate/mAMFGi and EpiSCs).

Real-Time PCR for chromatin immunoprecipitation (ChIP-qPCR): ChIP wasperformed using a commercially available Magna ChIP™ G kit (catalog#17-611, Millipore). Briefly, feeder-free cultured 1×10⁶ cells werefixed with 1% formaldehyde, lysed, and sonicated to obtain 200-500 bpDNA fragments conjugated with nucleosomes. The sonicated lysates wereimmunoprecipitated with anti-trimethyl-histone H3 lysine 4 (Millipore,Cat. #17-614, 3 ul/reaction) or anti-trimethyl-histone H3 lysine 27(Millipore, Cat. #17-622, 4 μg/reaction) that were in advance reactedwith secondary antibodies conjugated with magnetic beads. Afterincubation with each antibody for 24 hr, immunoprecipitants wererecovered and DNA fragments contained were purified by incubation withProteinase K. The DNA fragments were subjected to real-time PCR.Whole-cell lysates before incubation with antibodies were used as input.One microliter of DNA fragments from whole lysates andimmunoprecipitants were subject to real-time PCR reaction.Immunoprecipitants with normal Rabbit IgG served as negative controlsamples and showed no detectable background. Primer sequences werelisted in Table 2.

Table 2 Primers used for PCR Gene Forward (SEQ ID NO:)Reverse (SEQ ID NO:) For RT-PCR Blimp1 TCAGCCTCTTCCCTAGGTTGTATCAATCTTAAGGATCCATCGGTTCA (50) AC (51) Brachyury ATGCCAAAGAAAGAAACGAC (52)AGAGGCTGTAGAACATGATT 53) Cdx2 AGGCTGAGCCATGAGGAGTA (54)CGAGGTCCATAATTCCACTCA (55) Dax1 GTGGCAGGGCAGCATCCTCTACAACAAAAGAAGCGGTACA (57) (56) Esrrb CGCCATCAAATGCGAGTACATGAATCACCATCCAGGCACTCTG GC (58) (59) Fbxo15/ECAT3TAGATTCTTGGACTTCCGTTCA (60) ACCAAGGTCACCGCATCCAA (61) Fgf4CGTGGTGAGCATCTTCGGAGTGG CCTTCTTGGTCCGCCCGTTCTTA (62) (63) Fgf5CTGTACTGCAGAGTGGGCATCGG GACTTCTGCGAGGCTGCGACAG (64) G(65) GAPDHGTGTTCCTACCCCCAATGTGT (66) ATTGTCATACCAGGAAATGAGCT T (67) Gata6ACCTTATGGCGTAGAAATGCTGAG CTGAATACTTGAGGTCACTGTTC GGTG (68) TCGGG (69)Pecam1 GTCATGGCCATGGTCGAGTA (70) AGCAGGACAGGTCCAACAAC (71) Rex-1TGAAAGTGAGATTAGCCCCGAG GTCCCATCCCCTTCAATAGCAC (72) (73) StellaGAAACTCCTCAGAAGAAA (74) CTCTTGTTCTCCACAGGTAC (75) Stra8GCAACCAACCCAGTGATGATGG CATCTGGTCCAACAGCCTCAG (76) (77)For bisulfite-sectuencing PCR Fgf4 TTTAGGTTTTAAGAGTGTTGGGGAGTACAAAACAAAAACATCAAACC AAGAT (78) CATTCTAA (79) StellaATTTTGTGATTAGGGTTGGTTTAGA CCAAAACATCCTCTTCATCTTTC A (80) TTCT (81)Stella nest TTTTTGGAATTGGTTGGGATTG (82) CTTCTAAAAAATTTCAAAATCCTTCATT (83) For ChIP-Q PCR Stella GATCCAGCTGGTCTGAGCTA (84)GTGCAGGGATCATAGGAGTG (85)

Example 6 Characterization of Pluripotent Animal Cell that Replicatesand Maintains Pluripotency

To characterize the newly generated hiPSCs, real-time PCR was employedto analyze the gene expression of hiPSCs. Human ES cell line, Hues9 (hESfacility, Harvard University, http://mcb.harvard.edu/melton/hues/), wasused as a control. Real-time PCR analysis reveal that, as compared toHues9 cells, hiPSCs of the present invention express certain genes athigher levels, such as Gbx2 (5-fold), Dppa3 (2-fold) and Klf4(2.5-fold). These markers are also found to be highly expressed in mouseES cells, but not in mouse EpiSCs. The primers used in the real-time PCRassay are: Dppa3: 5′-CAACCTACATCCCAGGGTCT-3′ (SEQ ID NO:86);5′-TCAACGTCTCGGAGGAGATT-3′ (SEQ ID NO:87); Gbx2:5′-AAAGGCTTCCTGGCCAAAG-3′ (SEQ ID NO:88); 5′-TTGACTCGTCTTTCCCTTGC-3′(SEQ ID NO:89); Klf4: 5′-AGCCTAAATGATGGTGCTTGGT-3′ (SEQ ID NO:90);5′-TTGAAAACTTTGGCTTCCTTGTT-3′ (SEQ ID NO:91).

Using western-blot analysis, we have also analyze the expression ofE-cadherin in hiPSCs. The expression of E-cadherin in hiPSCs is twicethat of Hues9 human ES cells.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, databases, Genbank sequences,patents, and patent applications cited herein are hereby incorporated byreference.

1-57. (canceled)
 58. A method of manufacturing cells for therapeutic usecomprising culturing pluripotent cells through at least one celldivision in the presence of a sufficient amount of: (a) a compoundselected from one or more of a MEK inhibitor, an Erk inhibitor, a p38inhibitor, and an FGF receptor inhibitor; (b) a GSK3β inhibitor; and (c)nutrients; to allow for at least one cell division while maintainingcell pluripotency, and thereby obtaining cells for therapeutic use. 59.The method of claim 58, wherein the culturing step further comprisesculturing the cells in the presence of a Leukemia inhibitory factor(LIF).
 60. The method of claim 58, wherein the GSK3β inhibitor isCHIR99021.
 61. The method of claim 58, wherein the compound is a MEKinhibitor.
 62. The method of claim 61, wherein the MEK inhibitor isPD0325901.
 63. The method of claim 58, wherein the pluripotent cells arecultured through at least five cell divisions while maintaining cellpluripotency.
 64. The method of claim 58, further comprising introducinga heterologous nucleic acid into the pluripotent cells and culturing theresulting cells to allow for at least one additional cell division whilemaintaining pluripotency.
 65. The method of claim 58, wherein thepluripotent cells are rat, human, non-human primate, ovine, bovine,feline, canine, or porcine cells.
 66. A therapeutic compositioncomprising pluripotent animal cells for therapeutic use, wherein thepluripotent mammalian cells are obtained by a method comprisingculturing pluripotent cells through at least one cell passage in thepresence of a sufficient amount of: (a) a compound selected from one ormore of a MEK inhibitor and an FGF receptor inhibitor; and (b) a GSK3βinhibitor; to allow for at least one cell division while maintainingcell pluripotency.
 67. The therapeutic composition of claim 66, whereinthe pluripotent cells are further in the presence of Leukemia inhibitoryfactor (LIF).
 68. The therapeutic composition of claim 66, wherein theGSK3β inhibitor is CHIR99021.
 69. The therapeutic composition of claim66, wherein the compound is a MEK inhibitor.
 70. The therapeuticcomposition of claim 69, wherein the MEK inhibitor is PD0325901.
 71. Thetherapeutic composition of claim 66, wherein the pluripotent animalcells are human cells or rat cells.
 72. The therapeutic composition ofclaim 66, wherein the pluripotent animal cells are embryonic stem cells.